bluuebunny/crossref_metadata_2025 huggingface dataset:
Distribution of viruses in the Chesapeake Bay
Authors: Wommack K E , Hill R T , Kessel M , Russek-Cohen E , Colwell R R
Published: January 2020

High virus counts were found in water samples collected from the Chesapeake Bay. Viruses were enumerated by ultracentrifugation of water samples onto grids which were visualized by transmission electron microscopy. Virus counts in September 1990, April 1991, June 1991, August 1991, and October 1991 ranged between 2.6 x 10(6) and 1.4 x 10(8) viruses ml-1 with a mean of 2.5 x 10(7) viruses ml-1. Virus counts were usually at least three times higher than direct bacterial counts in corresponding samples. Virus counts in August and October were significantly higher than at the other sampling times, whereas bacterial counts were significantly lower at that time, yielding mean virus-to-bacterium ratios of 12.6 and 25.6, respectively. From analysis of morphology of the virus particles, it is concluded that a large proportion of the viruses are bacteriophages. The high virus counts obtained in this study suggest that viruses may be an important factor affecting bacterial populations in the Chesapeake Bay, with implications for gene transfer in natural aquatic bacterial populations and release of genetically engineered microorganisms to estuarine and coastal environments.
Identification of individual prokaryotic cells by using enzyme-labeled, rRNA-targeted oligonucleotide probes
Authors: Amann R I , Zarda B , Stahl D A , Schleifer K H
Published: January 2020

A method to microscopically detect and identify individual cells of members of the domains Bacteria and Archaea is presented. rRNA-targeted oligonucleotides were 5' end labeled with the enzyme horseradish peroxidase and used for whole-cell hybridization. Specifically bound probe was visualized by the enzymatic formation of an intracellular precipitate from the substrate diaminobenzidine. Permeation of the enzyme-labeled probe into whole fixed cells of gram-negative bacteria required their pretreatment with lysozyme-EDTA, whereas permeability of some archaebacterial cells was improved by addition of detergent to the hybridization buffer. Hitherto we had not achieved penetration of enzyme-labeled probe into gram-positive bacteria and yeast cells. This method should be a valuable tool for identification of suitable prokaryotic cells in environments with elevated background fluorescence or in situations in which an epifluorescence microscope is not available.
Detection of virulence factors in culturable Escherichia coli isolates from water samples by DNA probes and recovery of toxin-bearing strains in minimal o-nitrophenol-beta-D-galactopyranoside-4-methylumbelliferyl-beta-D-g luc uronide media
Authors: Martins M T , Rivera I G , Clark D L , Olson B H
Published: January 2020

A total of 449 Escherichia coli isolates in treated and raw water sources were submitted to DNA-DNA hybridization using seven different DNA probes to detect homology to sequences that code for Shiga-like toxins I and II; heat-stabile and heat-labile toxins, adherence factors EAF and eae, and the fimbrial antigen of entero-hemorrhagic E. coli. Fifty-nine (13%) of the isolates demonstrated homology with one or more specific DNA probes. More than 50% of the isolates in treated water were not recovered in MMO-4-methylumbelliferyl-beta-D-glucuronide media designed for detection of this indicator.
Glutamine synthetase and nitrogen cycling in colonies of the marine diazotrophic cyanobacteria Trichodesmium spp
Authors: Carpenter E J , Bergman B , Dawson R , Siddiqui P J , Söderbäck E , Capone D G
Published: January 2020

We examined freshly collected samples of the colonial planktonic cyanobacterium Trichodesmium thiebautii to determine the pathways of recently fixed N within and among trichomes. High concentrations of glutamate and glutamine were found in colonies. Glutamate and glutamine uptake rates and concentrations in cells were low in the early morning and increased in the late morning to reach maxima near midday; then uptake and concentration again fell to low values. This pattern followed that previously observed for T. thiebautii nitrogenase activity. Our results suggest that recently fixed nitrogen is incorporated into glutamine in the N2-fixing trichomes and may be passed as glutamate to non-N2-fixing trichomes. The high transport rates and concentrations of glutamate may explain the previously observed absence of appreciable uptake of NH4+, NO3-, or urea by Trichodesmium spp. Immunolocalization, Western blots (immunoblots), and enzymatic assays indicated that glutamine synthetase (GS) was present in all cells during both day and night. GS appeared to be primarily contained in cells of T. thiebautii rather than in associated bacteria or cyanobacteria. Double immunolabeling showed that cells with nitrogenase (Fe protein) contained levels of the GS protein that were twofold higher than those in cells with little or no nitrogenase. GS activity and the uptake of glutamine and glutamate dramatically decreased in the presence of the GS inhibitor methionine sulfoximine. Since no glutamate dehydrogenase activity was detected in this species, GS appears to be the primary enzyme responsible for NH3 incorporation.
Characterization of transcription initiation and termination signals of the proteinase genes of Lactococcus lactis Wg2 and enhancement of proteolysis in L. lactis
Authors: van der Vossen J M , Kodde J , Haandrikman A J , Venema G , Kok J
Published: January 2020

The transcription initiation signals of the prtP and prtM genes specifying the proteolytic activity of Lactococcus lactis subsp. cremoris Wg2 were mapped by primer extension. The strength of these promoters was analyzed with promoter-screening vector pGKV410, and they appeared to be weaker than previously isolated promoters of strain Wg2. In addition, a putative transcription terminator downstream of the prtP gene was characterized by using the terminator-screening vector pGKV259. The putative terminator decreased the transcription activity of lactococcal promoter P59 by approximately 70% in both Bacillus subtilis and L. lactis. Deletion of a part of the stem-loop structure of the terminator decreased the negative effect on transcription, indicating that the structure could indeed function as a terminator of transcription. The proteolytic activity of the lactococcal host was enhanced by placing the originally oppositely oriented prt genes in tandem and replacing the relatively weak promoters upstream of the prt genes with the stronger promoter, P32, from the chromosome of L. lactis Wg2.
Evaluation of the ability of primary selective enrichment to resuscitate heat-injured and freeze-injured Listeria monocytogenes cells
Authors: Budu-Amoako E , Toora S , Ablett R F , Smith J
Published: January 2020

Resuscitation rates of injured Listeria monocytogenes on conventional selective Listeria enrichment broth and nonselective Trypticase soy broth containing 0.6% yeast extract were compared. Cells were heated to 60 degrees C for 5 min or frozen at -20 degrees C for 7 days. Inoculation of Trypticase soy broth-yeast extract with the stressed cells resulted in growth that was superior to that in Listeria enrichment broth. Injured cells were fully recovered at 6 to 8 h.
Identification and ecology of bacterial communities associated with necroses of three cactus species
Authors: Foster J L , Fogleman J C
Published: January 2020

To compare the bacterial communities residing in necrotic tissues of columnar cacti of the Sonoran Desert, isolates from 39 organ pipe, 19 saguaro, and 16 senita cacti were obtained. The isolates were clustered into 28 conspecific groups on the basis of their fatty acid profiles. The distributions of the individual bacterial isolates varied among cactus species. Seven of the 28 species groups were unique to a particular cactus species, whereas 8 species groups were found in all three cacti. The effective number of bacterial species for each cactus species was positively correlated with both the chemical complexity and glucose concentration of the plant tissues. The effective number of bacterial species and bacterial distribution patterns were compared with those known for communities of cactophilic yeasts. The observed bacterial distribution patterns are most likely due to differences in the chemical compositions of the three cactus species.
Comparison of the post-Chernobyl 137Cs contamination of mushrooms from eastern Europe, Sweden, and North America
Authors: Smith M L , Taylor H W , Sharma H D
Published: January 2020

A comparison was made of 134Cs and 137Cs contamination in fungi from eastern Europe and eastern North America. Mean activities of 25 Ukrainian, 6 Swedish, and 10 North American collections were 4,660, 9,750, and 205 Bq/kg (dry weight), respectively. Additional measurements were made on samples from the Moscow, southern Belarus, and Yugoslavia/Bulgaria regions. Activity values were found to vary by several orders of magnitude within all geographic areas, even for the same mushroom species. Significantly higher specific activities were observed in mycorrhizal species than in saprophytic and parasitic fungi. Unfortunately, many of the European mycorrhizal species considered as prized edibles contained unacceptably high levels of 137Cs (> 1,000 Bq/kg [dry weight]) and should be used sparingly as food. By contrast, no mushrooms collected in Ontario or northern Michigan exceeded 1,000 Bq of 137Cs per kg (dry weight). The excessive 137Cs contamination was evident in mushrooms from areas that had substantial fallout from the 1986 accident in reactor 4 at the Chernobyl nuclear power station. However, observations suggest that about 20% of the 137Cs in eastern Europe (Moscow area, Belarus, and Ukraine) is of non-Chernobyl origin.
Detection and distribution of rotavirus in raw sewage and creeks in São Paulo, Brazil
Authors: Mehnert D U , Stewien K E
Published: January 2020

Rotaviruses were concentrated from 8-liter samples of raw domestic sewage and sewage-polluted creek water by adsorption to and elution from positively charged microporous filters (Zeta Plus 60S), followed by ultracentrifugation of the filter eluates. Indirect immunofluorescence and direct immunoperoxidase methods allowed detection and enumeration of rotavirus in 6 (20.6%) of 29 sewage samples and in 19 (34.5%) of 55 creek water samples. Levels of rotaviruses ranged from < 3 to 63 focus-forming units (FFU)/liter, and the geometric means were 2.2 FFU/liter in sewage, 2.9 FFU/liter at creek Tremembé, and 2.6 FFU/liter at creek Pirajussara. Wastewater samples examined during autumn and winter months showed a higher rate positivity for rotavirus than those collected in spring and summer, corresponding to the seasonal variation of rotaviral diarrhea in the city of São Paulo.
Evidence Regarding the UV Sunscreen Role of a Mycosporine-Like Compound in the Cyanobacterium
Gloeocapsa
sp
Authors: Garcia-Pichel Ferran , Wingard Christopher E. , Castenholz Richard W.
Published: January 2020

The UV sunscreen role commonly ascribed to mycosporine-like amino acids (MAAs) was investigated with an isolate of the terrestrial cyanobacterium
Gloeocapsa
sp. strain C-90-Cal-G.(2), which accumulates intracellularly an MAA with absorbance maximum at 326 nm but produces no extracellular sunscreen compound (i.e., scytonemin). The intracellular concentrations of MAA achieved were directly related to the intensity of the UV radiation (maximum at 320 nm) received by the cells. However, the presence of high concentrations of MAA was not necessary for the physiological acclimation of the cultures to UV radiation. The measured sunscreen factor due to MAA in single cells was 0.3 (the MAA prevented 3 out of 10 photons from hitting potential cytoplasmic targets). High contents of MAA in the cells correlated with increased resistance to UV radiation. However, when resistance was gauged under conditions of desiccation, with inoperative physiological photoprotective and repair mechanisms, cells with high MAA specific contents were only 20 to 25% more resistant. Although UV radiation centered around both 320 and 365 nm resulted in chlorophyll
a
photobleaching and photoinhibition of photosynthesis, the difference in sensitivity correlated with MAA accumulation occurred only at 320 nm (absorbed by MAA) and not at 365 nm (not absorbed by MAA). This difference represents the maximal protection ascribable to the presence of MAA for single cells, i.e., if one does not consider the enhancing effects of colony formation on protection by sunscreens.
Adaptation and growth of Serratia marcescens in contact lens disinfectant solutions containing chlorhexidine gluconate
Authors: Gandhi P A , Sawant A D , Wilson L A , Ahearn D G
Published: January 2020

Serratia marcescens (11 of 12 strains) demonstrated an ability to grow in certain chlorhexidine-based disinfecting solutions recommended for rigid gas-permeable contact lenses. For a representative strain, cells that were grown in nutrient-rich medium, washed, and inoculated into disinfecting solution went into a nonrecoverable phase within 24 h. However, after 4 days, cells that had the ability to grow in the disinfectant (doubling time, g = 5.7 h) emerged. Solutions supporting growth of S. marcescens were filter sterilized. These solutions, even after removal of the cells, showed bactericidal activity against Pseudomonas aeruginosa and a biphasic survival curve when rechallenged with S. marcescens. Adaptation to chlorhexidine by S. marcescens was not observed in solutions formulated with borate ions. For chlorhexidine-adapted cells, the MIC of chlorhexidine in saline was eightfold higher than that for unadapted cells. Cells adapted to chlorhexidine showed alterations in the proteins of the outer membrane and increased adherence to polyethylene. Cells adapted to chlorhexidine persisted or grew in several other contact lens solutions with different antimicrobial agents, including benzalkonium chloride.
Lactic Acid Production in a Mixed-Culture Biofilm Reactor
Authors: Demirci Ali , Pometto Anthony L. , Johnson Kenneth E.
Published: January 2020

Novel solid supports, consisting of polypropylene blended with various agricultural materials (pp composite), were evaluated as supports for pure- and mixed-culture continuous lactic acid fermentations in biofilm reactors.
Streptomyces viridosporus
T7A (ATCC 39115) was used to form a biofilm, and
Lactobacillus casei
subsp.
rhamnosus
(ATCC 11443) was used for lactic acid production. For mixed-culture fermentations, a 15-day continuous fermentation of
S. viridosporus
was performed initially to establish the biofilm. The culture medium was then inoculated with
L. casei
subsp.
rhamnosus.
For pure-culture fermentation,
L. casei
subsp.
rhamnosus
was inoculated directly into the reactors containing sterile pp composite chips. The biofilm reactors containing various pp composite chips were compared with a biofilm reactor containing pure polypropylene chips and with a reactor containing a suspension culture. Continuous fermentation was started, and each flow rate (0.06 to 1.92 ml/min) was held constant for 24 h; steady state was achieved after 10 h. Lactic acid production was determined throughout the 24-h period by high-performance liquid chromatography. Production rates that were two to five times faster than those of the suspension culture (control) were observed for the pure- and mixed-culture bioreactors. Both lactic acid production rates and lactic acid concentrations in the culture medium were consistently higher in mixed-culture than in pure-culture fermentations. Biofilm formation on the chips was detected at harvest by chip clumping and Gram staining.
Monitoring airborne fungal spores in an experimental indoor environment to evaluate sampling methods and the effects of human activity on air sampling
Authors: Buttner M P , Stetzenbach L D
Published: January 2020

Aerobiological monitoring was conducted in an experimental room to aid in the development of standardized sampling protocols for airborne microorganisms in the indoor environment. The objectives of this research were to evaluate the relative efficiencies of selected sampling methods for the retrieval of airborne fungal spores and to determine the effect of human activity on air sampling. Dry aerosols containing known concentrations of Penicillium chrysogenum spores were generated, and air samples were taken by using Andersen six-stage, Surface Air System, Burkard, and depositional samplers. The Andersen and Burkard samplers retrieved the highest numbers of spores compared with the measurement standard, an aerodynamic particle sizer located inside the room. Data from paired samplers demonstrated that the Andersen sampler had the highest levels of sensitivity and repeatability. With a carpet as the source of P. chrysogenum spores, the effects of human activity (walking or vacuuming near the sampling site) on air sampling were also examined. Air samples were taken under undisturbed conditions and after human activity in the room. Human activity resulted in retrieval of significantly higher concentrations of airborne spores. Surface sampling of the carpet revealed moderate to heavy contamination despite relatively low airborne counts. Therefore, in certain situations, air sampling without concomitant surface sampling may not adequately reflect the level of microbial contamination in indoor environments.
Production of (
R
)-3-Chloro-1,2-Propanediol from Prochiral 1,3-Dichloro-2-Propanol by
Corynebacterium
sp. Strain N-1074
Authors: Nakamura Tetsuji , Yu Fujio , Mizunashi Wataru , Watanabe Ichiro
Published: January 2020

The production of (
R
)-3-chloro-1,2-propanediol [(
R
)-MCP] from prochiral 1,3-dichloro-2-propanol (DCP) was examined with a bacterial strain identified as a
Corynebacterium
strain. The addition of glycerol as a carbon source or some chlorinated alcohols to a medium was effective for the induction of activity catalyzing the transformation of DCP into MCP. The optimum pH for (
R
)-MCP production by the resting cell reaction was around 8.0. The optical purity of (
R
)-MCP formed was improved by keeping the level of DCP in the reaction mixture at a low concentration. (
R
)-MCP was obtained from 77.5 mM DCP with a 97.3% molar conversion yield and an 83.8% enantiomeric excess of its optical purity by periodic feeding of the substrate.
Molecular analysis of the rolling-circle replicating plasmid pA1 of Lactobacillus plantarum A112
Authors: Vujcic M , Topisirovic L
Published: January 2020

Lactobacillus plantarum A112 has four different plasmids. Plus-origin-specific probes were used to determine that the smallest, cryptic plasmid, pA1 (2,820 bp), showed homology to the pE194 plasmid family. This subclass of plasmids uses the rolling-circle mode of replication. Subsequent analysis of plasmid pA1 demonstrated that it generates single-stranded DNA intermediates, and sequence analysis revealed that it contains three putative open reading frames (ORFs): ORF1, ORF2, and ORF3, which could encode proteins designated RepA (47 amino acids [aa]) and RepB (196 aa) and a protein of 103 aa, respectively. Two of these proteins, RepA (5.6 kDa) and RepB (26 kDa), were identified in in vitro transcription translation assays. The RepA protein contains a characteristic alpha-helix-turn-alpha-helix motif typical of DNA-binding proteins that act as DNA-binding repressors. The RepB protein shows a significant similarity with replication initiation proteins of the pE194 family of plasmids that use the rolling-circle mode of replication. Plasmid pA1 is able to replicate in Escherichia coli and Lactobacillus lactis subsp. lactis as well as in other L. plantarum strains.
Growth, copper-tolerant cells, and extracellular protein production in copper-stressed chemostat cultures of Vibrio alginolyticus
Authors: Gordon A S , Harwood V J , Sayyar S
Published: January 2020

The influence of elevated copper concentrations on cell numbers and extracellular protein production was investigated in chemostat cultures of Vibrio alginolyticus. High (20 microM) copper in the medium reservoir resulted in a dramatic drop in cell numbers which was overcome with time. The copper-stressed cultures established a new equilibrium cell concentration slightly (ca. 20%) lower than control cultures. Copper-stressed chemostat populations contained an increased number of copper-resistant cells, but these averaged only 26% of the copper-adapted population. Previously copper-stressed populations exhibited resistance to a second challenge with copper. Proteins with properties identical to those of copper-induced, copper-binding proteins (CuBPs) observed in batch cultures of V. alginolyticus were observed in the supernatants of copper-stressed chemostat cultures and not in controls. CuBPs from batch and chemostat cultures were identical in terms of their induction by copper, molecular weight, and retention volumes on both immobilized copper ion-affinity chromatography and reverse-phase high-performance liquid chromatography columns. The concentration of CuBP in the chemostat was dependent on copper concentration in the medium reservoir. Either one or two forms of CuBP were observed in various analyses from both batch and chemostat cultures. Gel-to-gel variability was implicated as a factor determining whether one or two forms were resolved in a given analysis.(ABSTRACT TRUNCATED AT 250 WORDS)
Effects of Calcium on Sugar Transport in
Azotobacter vinelandii
Authors: Wong T. Y.
Published: January 2020

A fast and environmentally safe procedure was used to study sugar uptake by
Azotobacter vinelandii.
Transport experiments were performed in a 24-well plate and aerated by rapid oscillatory vibration. Samples were washed by centrifugation and dissolved in biodegradable scintillation cocktail for counting. At cell concentrations up to 6 × 10
8
cells per ml, the uptake of sucrose was a function of time and was proportional to the cell concentration. This modified uptake assay was used to test the effect of cations on sugar uptake in
A. vinelandii.
Results showed that Ca
2+
at 1 to 2 mM stimulated sucrose uptake by decreasing the apparent

K
m

of sucrose transport. Higher Ca
2+
concentrations inhibited sucrose uptake in this organism.
Characterization of Loosely Associated Material from the Cell Surface of
Lactococcus lactis
subsp.
cremoris
E8 and Its Phage-Resistant Variant Strain 398
Authors: Gopal Pramod K. , Crow Vaughan L.
Published: January 2020

Loosely associated material (LAM) was isolated by gentle extraction procedures from the cell surface of
Lactococcus lactis
subsp.
cremoris
E8 and its phage-resistant variant strain 398. LAM from both strains was chemically characterized, and its role in the adsorption of three small isometric bacteriophages, φ 618, φ 833, and φ 852, to the cell surface of the two strains was investigated. The phage-resistant strain (strain 398) produced LAM which differed significantly from the material produced by the parent strain. The total yield of LAM from strain 398 was two- to threefold higher than that from strain E8, and the material contained fivefold more rhamnose and twofold more galactose. Polyacrylamide gel electrophoretic analysis showed that LAM from strain 398 lacked a 21-kDa protein which was present in LAM from the parent strain. Inhibition studies of phage binding by using isolated LAM from two strains showed that although LAM from strain E8 reduced the titer of φ 618 and φ 852 by 53 and 82% respectively, LAM from strain 398 had no effect on the plaque-forming ability of any of the three phages tested. Treatment of LAM from strain E8 with sodium metaperiodate destroyed its ability to bind with φ 618 and φ 852. Phenotypically, strain 398 differed from its parent strain E8 in that it was more prone to cell lysis and required an osmotically adjusted buffer system for the extraction of LAM.
Bacterial aerosol emission rates from municipal wastewater aeration tanks
Authors: Sawyer B , Elenbogen G , Rao K C , O'Brien P , Zenz D R , Lue-Hing C
Published: January 2020

In this report we describe the results of a study conducted to determine the rates of bacterial aerosol emission from the surfaces of the aeration tanks of the Metropolitan Water Reclamation District of Greater Chicago John E. Egan Water Reclamation Plant. This study was accomplished by conducting test runs in which Andersen six-stage viable samplers were used to collect bacterial aerosol samples inside a walled tower positioned above an aeration tank liquid surface at the John E. Egan Water Reclamation Plant. The samples were analyzed for standard plate counts (SPC), total coliforms (TC), fecal coliforms, and fecal streptococci. Two methods of calculation were used to estimate the bacterial emission rate. The first method was a conventional stack emission rate calculation method in which the measured air concentration of bacteria was multiplied by the air flow rate emanating from the aeration tanks. The second method was a more empirical method in which an attempt was made to measure all of the bacteria emanating from an isolated area (0.37 m2) of the aeration tank surface over time. The data from six test runs were used to determine bacterial emission rates by both calculation methods. As determined by the conventional calculation method, the average SPC emission rate was 1.61 SPC/m2/s (range, 0.66 to 2.65 SPC/m2/s). As determined by the empirical calculation method, the average SPC emission rate was 2.18 SPC/m2/s (range, 1.25 to 2.66 SPC/m2/s). For TC, the average emission rate was 0.20 TC/m2/s (range, 0.02 to 0.40 TC/m2/s) when the conventional calculation method was used and 0.27 TC/m2/s (range, 0.04 to 0.53 TC/m2/s) when the empirical calculation method was used.(ABSTRACT TRUNCATED AT 250 WORDS)
Two Uptake Systems for Fructose in
Lactococcus lactis
subsp.
cremoris
FD1 Produce Glycolytic and Gluconeogenic Fructose Phosphates and Induce Oscillations in Growth and Lactic Acid Formation
Authors: Benthin Stig , Nielsen Jens , Villadsen John
Published: January 2020

Fructose transport in lactococci is mediated by two phosphotransferase systems (PTS). The constitutive mannose PTS has a broad specificity and may be used for uptake of fructose with a fructose saturation constant (
K
Fru
) of 0.89 mM, giving intracellular fructose 6-phosphate. The inducible fructose PTS has a very small saturation constant (
K
Fru
, <17 μM), and the fructose 1-phosphate produced enters the Embden-Meyerhof-Parnas (EMP) pathway as fructose 1,6-diphosphate. Growth in batch cultures of
Lactococcus lactis
subsp.
cremoris
FD1 in a yeast extract medium with fructose as the only sugar is poor both with respect to specific growth rate and biomass yield, whereas the specific lactic acid production rate is higher than those in similar fermentations on other sugars metabolized via the EMP pathway, e.g., glucose. In fructose-limited chemostat cultures, the biomass concentration exhibits a strong correlation with the dilution rate, and starting a continuous culture at the end of a batch fermentation leads to large and persistent oscillations in the biomass concentration and specific lactic acid production rate. Two proposed mechanisms underlying this strange growth pattern follow. (i) Fructose transported via the fructose PTS cannot be converted into essential biomass precursors (glucose 6-phosphate or fructose 6-phosphate), because
L. lactis
subsp.
cremoris
FD1 is devoid of fructose 1,6-diphosphatase activity. (ii) The fructose PTS apparently produces a metabolite (presumably fructose 1-phosphate) which exerts catabolite repression of both mannose PTS and lactose PTS. Since the repressed mannose PTS and lactose PTS are shown to have identical maximum molar transport rates, the results indicate that it is the general PTS proteins which are repressed.
Effect of monensin on the specific activity of ammonia production by ruminal bacteria and disappearance of amino nitrogen from the rumen
Authors: Yang C M , Russell J B
Published: January 2020

When unadapted mixed ruminal bacteria (312 mg of protein per liter) were treated with monensin (5 mM) in vitro, the rates of ammonia production from enzymatic digests of casein, gelatin, and soy protein (0.5 g of N per liter) were decreased from 46 +/- 2 to 24 +/- 1, 20 +/- 1 to 7 +/- 1, and 40 +/- 2 to 18 +/- 2 nmol/mg of protein per min, respectively. Monensin also caused a decrease in ammonia production in vivo. Nonlactating dairy cows which were fed 0.56 kg of timothy hay 12 times per day had a steady-state ruminal ammonia concentration of 2.7 +/- 0.1 mM, and the ammonia concentration decreased to 1.2 +/- 0.2 mM when monensin (350 mg/day) was added to the diet. The decrease in ammonia production was associated with a 10-fold reduction (4.1 x 10(6) versus 4.2 x 10(5)/ml) in the most probable number of ammonia-producing ruminal bacteria that could use protein hydrolysate as an energy source. Monensin had little effect on the most probable number of carbohydrate-utilizing ruminal bacteria (6.5 versus 7.0 x 10(8)/ml). The addition of protein hydrolysates (560 g) to the rumen caused a rapid increase in the ammonia concentration, but this increase was at least 30% lower when the animals were fed monensin.(ABSTRACT TRUNCATED AT 250 WORDS)
Survival of Denitrifiers in Nitrate-Free, Anaerobic Environments
Authors: Jørgensen Kirsten S. , Tiedje James M.
Published: January 2020

Experiments were undertaken to explain the occurrence of a high denitrification capacity in anaerobic, NO
3
-
-free habitats. Deep layers of freshwater sediments that were buried more than 40 years ago and digested sludge were the habitats studied. The denitrifier populations were 3.1 × 10
3
and 3.1 × 10
5
cells cm
-3
in deep sediments from a river and lake, respectively, and 5.3 × 10
6
cells cm
-3
in digested sludge. The denitrification capacities of the samples reflected the population densities. Strict anaerobic procedures were used to obtain the predominant isolates that would grow on anaerobic medium with NO
3
-
. All strict anaerobes isolated failed to denitrify. All isolates that denitrified were aerobic, gram-negative bacteria, particularly species of
Pseudomonas
and
Alcaligenes
. No detectable growth was observed when these strains were incubated with electron acceptors other than NO
3
-
or O
2
. When representative isolates were added to sterile, O
2
- and NO
3
-
-free porewater from their original locations at their natural densities (10
5
cells cm
-3
), no change in viable population was noted over 3 months of incubation. Metabolic activity was demonstrated in these cells by slow formation of formazan granules when exposed to tetrazolium and by observation of motile cells. When [
14
C]glucose was added to cell suspensions of the pseudomonads that had been starved for 3 months without electron acceptors (O
2
or NO
3
-
),
14
C-labeled products, including cell biomass,
14
CO
2
, and fermentation products, were produced. The high denitrification capacity of these anaerobic environments appears to be due to conventional respiratory denitrifiers. These organisms have the capacity for long-term survival without O
2
or NO
3
-
and appear to be capable of providing for their maintenance by carrying on a low level of fermentation.
Anaerobic degradation of nitrilotriacetate (NTA) in a denitrifying bacterium: purification and characterization of the NTA dehydrogenase-nitrate reductase enzyme complex
Authors: Jenal-Wanner U , Egli T
Published: January 2020

The initial step in the anoxic metabolism of nitrilotriacetate (NTA) was investigated in a denitrifying member of the gamma subgroup of the Proteobacteria. In membrane-free cell extracts, the first step of NTA oxidation was catalyzed by a protein complex consisting of two enzymes, NTA dehydrogenase (NTADH) and nitrate reductase (NtR). The products formed were iminodiacetate and glyoxylate. Electrons derived from the oxidation of NTA were transferred to nitrate only via the artificial dye phenazine methosulfate, and nitrate was stoichiometrically reduced to nitrite. NTADH activity could be measured only in the presence of NtrR and vice versa. The NTADH-NtrR enzyme complex was purified and characterized. NTADH and NtrR were both alpha 2 dimers and had molecular weights of 170,000 and 105,000, respectively. NTADH contained covalently bound flavin cofactor, and NtrR contained a type b cytochrome. Optimum NTA-oxidizing activity was achieved at a molar ratio of NTADH to NtrR of approximately 1:1. So far, NTA is the only known substrate for NTADH. This is the first report of a redox enzyme complex catalyzing the oxidation of a substrate and concomitantly reducing nitrate.
Comparison of ATP and Ergosterol as Indicators of Fungal Biomass Associated with Decomposing Leaves in Streams
Authors: Suberkropp K. , Gessner M. O. , Chauvet E.
Published: January 2020

ATP and ergosterol were compared as indicators of fungal biomass associated with leaves decomposing in laboratory microcosms and streams. In all studies, the sporulation rates of the fungi colonizing leaves were also determined to compare patterns of fungal reproductive activity with patterns of mycelial growth. During leaf degradation, ATP concentrations exhibited significant, positive correlations with ergosterol concentrations in the laboratory and when leaves had been air dried prior to being submerged in a stream. However, when freshly shed leaves were submerged in a stream, concentrations of ATP and ergosterol were negatively correlated during degradation. This appeared to be due to the persistence of leaf-derived ATP in freshly shed leaves during the first 1 to 2 weeks in the stream. Estimates of fungal biomass from ergosterol concentrations of leaf litter were one to three times those calculated from ATP concentrations. ATP, ergosterol, and sporulation data generally provided similar information about the fungi associated with decomposing leaves in streams during periods when fungi were growing. Ergosterol concentrations provide a more accurate indication of fungal biomass in situations in which other organisms make significant contributions to ATP pools.
Use of polymerase chain reaction and electroporation of Escherichia coli to monitor the persistence of extracellular plasmid DNA introduced into natural soils
Authors: Romanowski G , Lorenz M G , Wackernagel W
Published: January 2020

A modified protocol for DNA amplification by polymerase chain reaction (PCR) coupled with laser densitometric determination of the amount of PCR products, which allowed quantitation of target sequence numbers in soil extracts, was developed. The method was applied to monitor target loss during incubation of purified plasmid DNA in natural nonsterile soils. It revealed soil-specific kinetics of target loss. After 60 days, 0.2, 0.05, and 0.01% of the initially added nahA genes on plasmids were detectable by PCR in a loamy sand soil, a clay soil, and a silty clay soil, respectively. Electroporation of Escherichia coli was used in parallel to quantitate plasmid molecules in soil extracts by their transforming activity. It was found that transformation by electroporation was about 20 times more efficient and much less inhibited by constituents of soil extracts than transformation of Ca(2+)-treated cells (G. Romanowski, M.G. Lorenz, G. Sayler, and W. Wackernagel, Appl. Environ. Microbiol. 58:3012-3019, 1992). By electroporation, greater than 10,000-fold plasmid loss was monitored in nonsterile soils. Transforming activity was found up to 60 days after inoculation of the soils. The studies indicate that PCR and electroporation are sensitive methods for monitoring the persistence of extracellular plasmid DNA in soil. It is proposed that plasmid transformation by electroporation can be used for the monitoring in soil and other environments of genetically engineered organisms with recombinant plasmids. The data suggest that genetic material may persist in soil for weeks and even for months after its release from cells.
Differentiation of Lactobacillus strains by ribotyping
Authors: Rodtong S , Tannock G W
Published: January 2020

Fifty-four lactobacillus strains were differentiated by ribotyping. The stability of ribotypes characteristic of four strains of lactobacilli inhabiting the digestive tract of mice was investigated. One of four isolates of Lactobacillus delbrueckii GT21, which had been associated with mice for 22 months, had an altered ribotype.
Simple method of concentrating enteroviruses and hepatitis A virus from sewage and ocean water for rapid detection by reverse transcriptase-polymerase chain reaction
Authors: Tsai Y L , Sobsey M D , Sangermano L R , Palmer C J
Published: January 2020

A rapid and simple method was developed to detect enteroviruses and hepatitis A virus (HAV) in sewage and ocean water. Sewage samples were concentrated by Centriprep-100 and Centricon-100 at 1,000 x g. Samples collected from estuary and near-shore surf zone ocean water in Southern California were concentrated by vortex flow filtration and microconcentration. Reverse transcriptase-polymerase chain reaction (RT-PCR), with enterovirus primers or HAV capsid-specific primers, was used to detect enteroviruses or HAV in all concentrated samples. A nonradioactive internal probe was used to confirm the amplified products. Results of seeding experiments indicated that at 4 degrees C, HAV was more persistent than poliovirus in seawater and both HAV and poliovirus persisted longer at 4 degrees C than at 25 degrees C. RT-PCR was at least 500-fold more sensitive than cell culture. Results were obtained within 5 h by RT-PCR, in contrast with the 5 days to 3 weeks required for cell culture.
Polymerase chain reaction detection of nonviable bacterial pathogens
Authors: Josephson K L , Gerba C P , Pepper I L
Published: January 2020

Polymerase chain reaction (PCR) methodologies for detection of pathogens in environmental samples are currently available. However, positive amplification products for any set of primers only signal that the appropriate target nucleic acid sequences were present in the sample. The presence of the amplification products does not imply that the target organisms were viable. Here we show that PCR will detect nonviable cells, as long as intact target nucleic acid sequences are available. In an environmental water sample, nucleic acids degraded quickly and were not detectable by PCR after 3 weeks even when stored at 4 degrees C. However, these data show that there is a window of opportunity for PCR analyses to result in false positives with respect to viable cells. We further show that care must be taken in the way samples are stored for future PCR amplifications and that filter sterilization of media is not acceptable for long-term preservation of samples for PCR.
Direct plating procedure for enumerating Vibrio vulnificus in oysters (Crassostrea virginica)
Authors: Miceli G A , Watkins W D , Rippey S R
Published: January 2020

A procedure for enumerating and identifying Vibrio vulnificus in oysters was developed and evaluated. This method consists of growth on a direct plating medium (VVE medium) for isolating the organism from shellfish tissues, followed by biochemical tests for differentiating and identifying presumptively positive isolates. Densities of V. vulnificus are reliably obtained in 2 to 4 days, and as few as 10 culturable cells per 100 g can be identified. The procedure was evaluated by using a DNA probe technique specific for the cytotoxin-hemolysin gene of V. vulnificus and gas chromatographic analysis of the fatty acid contents of positive isolates. Only 3.2 and 0.4% of the isolates gave false-positive and false-negative results, respectively. The average level of recovery on VVE medium for 33 strains, including both clinical and environmental isolates, was 92% of the level of recovery obtained with brain heart infusion agar supplemented with 1% NaCl. The densities of V. vulnificus in oyster homogenates and individual oysters harvested from gulf and Atlantic coastal waters revealed that seasonally high levels occurred. The VVE medium procedure facilitated enumeration of this pathogen in molluscan shellfish and had a distinct advantage over the widely used most-probable-number procedure for V. vulnificus enumeration, which requires 5 to 7 days and often gives improbable and imprecise results.
Diversity of cell envelope proteinase specificity among strains of Lactococcus lactis and its relationship to charge characteristics of the substrate-binding region
Authors: Exterkate F A , Alting A C , Bruinenberg P G
Published: January 2020

The biochemical and genetical diversity of the subtilisin-like cell envelope proteinase (CEP) among Lactococcus lactis strains was investigated. The specificities of the proteinases of 16 strains toward the important cheese peptide alpha s1-casein fragment 1 to 23 and toward two differently charged chromophoric peptides have been determined. On the basis of the results, these strains could be classified into seven groups. The contribution to the specificity of specific residues in the large C-terminal segment, which differentiates this proteinase from most other members of the subtilisin family, was established with hybrid proteinases, even in the case of the small substrates. These remote residues and the subtilisin-like substrate-binding region are therefore assumed to be spatially close to each other and together constitute most of the binding region of CEP. DNA sequence analysis of fragments of the gene (prtP) encoding segments of the proteinase which contain the relevant residues of the substrate-binding region shows that among the strains studied, this binding region is the most negatively charged in the CEP group represented by strain HP and the positively charged in the CEP group represented by strains AM1 and SK11. Consequently, these two proteinase groups show the most divergent specificities. Each of the proteinases of the other groups shows a different intermediate specificity which in part is the reflection of an intermediate charge in the binding region. However, the results suggest that amino acid residues outside the segments known to be part of the CEP-binding region also contribute to specificity.(ABSTRACT TRUNCATED AT 250 WORDS)
Production of
Rhizobium
Inoculants for
Lupinus nootkatensis
on Nutrient-Supplemented Pumice
Authors: Einarsson Sigurbjorn , Gudmundsson Jon , Sverrisson Halldor , Kristjansson Jakob K. , Runolfsson Sveinn
Published: January 2020

The use of the legume
Lupinus nootkatensis
as a pioneer plant to fight soil erosion and to reclaim eroded soils in Iceland has been under development for a few years. Production of a robust, low-cost bacterial inoculant was therefore a prerequisite for the extended use of this plant. Volcanic pumice is a naturally expanded mineral which is available in vast amounts in Iceland. It was tested as a carrier for solid fermentation of
Rhizobium lupini.
Nutrient-supplemented pumice containing a small percentage of peat and diatomaceous earth and kept in sterile plastic bags promoted good growth of the bacteria. Viable-colony counts remained stable at 10
8
to 10
9
/g for at least 35 weeks when the carrier was stored at 22°C. The pumice-based inoculant had good storage and handling properties and could be mixed directly with the seeds during the sowing process. When seeds of
L. nootkatensis
were sown manually into nutrient-poor eroded sandy soils, about 56% of the first-year plants were successfully nodulated.
Comparison of Giardia lamblia and Giardia muris cyst inactivation by ozone
Authors: Finch G R , Black E K , Labatiuk C W , Gyürék L , Belosevic M
Published: January 2020

Inactivation of Giardia lamblia and Giardia muris cysts was compared by using an ozone demand-free 0.05 M phosphate buffer in bench-scale batch reactors at 22 degrees C. Ozone was added to each trial from a concentrated stock solution for contact times of 2 and 5 min. The viability of the control and treated cysts was evaluated by using the C3H/HeN mouse and Mongolian gerbil models for G. muris and G. lamblia, respectively. The resistance of G. lamblia to ozone was not significantly different from that of G. muris under the study conditions, contrary to previously reported data that suggested G. lamblia was significantly more sensitive to ozone than G. muris was. The simple Ct value for 2 log unit inactivation of G. lamblia was 2.4 times higher than the Ct value recommended by the Surface Water Treatment Rule.
A simple, sensitive, and rapid method for detecting seed contaminated with highly virulent Leptosphaeria maculans
Authors: Taylor J L
Published: January 2020

A primer-directed DNA amplification polymerase chain reaction assay for detection of seed contaminated with highly virulent Leptosphaeria maculans was developed. The primers were derived from a 5,238-bp repetitive sequence present only in the highly virulent isolates of the fungus. A procedure for isolating DNA from organisms infesting germinating seed was also developed. Seeds were added to liquid fungal minimal medium, and the culture was incubated for 3 days at room temperature with shaking. The organisms were collected from the cultures by centrifugation and lysed with a combination of sodium dodecyl sulfate and proteinase K. The DNA was extracted with organic solvents and with a high-salt-cetyltrimethylammonium bromide solution. It was also precipitated with a low-salt-cetyltrimethylammonium bromide solution. The extensive treatments used for minimizing polysaccharide contamination greatly improved the reliability of the assay. The minimum contamination level (2 of 1,000 seeds) that was tested was successfully detected with this DNA isolation procedure. The reliability of the assay was 96% at the 1 to 2% level of seed contamination. The described method is less laborious and requires only 4 to 5 days for completion in comparison to the 11 to 22 days required for the currently employed methods. In addition, large sample sizes can be easily handled, thus reducing the probability of contaminated seed escaping detection.
A substrate-dependent biological containment system for Pseudomonas putida based on the Escherichia coli gef gene
Authors: Jensen L B , Ramos J L , Kaneva Z , Molin S
Published: January 2020

A model substrate-dependent suicide system to biologically contain Pseudomonas putida KT2440 is reported. The system consists of two elements. One element carries a fusion between a synthetic lac promoter (PA1-04/03) and the gef gene, which encodes a killing function. This element is contained within a transposaseless mini-Tn5 transposon so that it can be integrated at random locations on the Pseudomonas chromosome. The second element, harbored by plasmid pCC102, is designed to control the first and bears a fusion between the promoter of the P. putida TOL plasmid-encoded meta-cleavage pathway operon (Pm) and the lacI gene, encoding the Lac repressor, plus xylS2, coding for a positive regulator of Pm. In liquid culture under optimal growth conditions and in sterile and nonsterile soil microcosms, P. putida KT2440 (pWWO) bearing the containment system behaves as designed. In the presence of a XylS effector, such as m-methylbenzoate, the LacI protein is synthesized, preventing the expression of the killing function. In the absence of effectors, expression of the PA1-04/03::gef cassette is no longer prevented and a high rate of cell killing is observed. Fluctuation test analyses revealed that mutants resistant to cell killing arise at a frequency of around 10(-5) to 10(-6) per cell per generation. Mutations are linked to the killing element rather than to the regulatory one. In bacteria bearing two copies of the killing cassette, the rate of appearance of mutants resistant to killing decreased to as low as 10(-8) per cell per generation.