bluuebunny/crossref_metadata_2025 huggingface dataset:
Precise Detection and Tracing of
Trichoderma hamatum
382 in Compost-Amended Potting Mixes by Using Molecular Markers
Authors: Abbasi Pervaiz A. , Miller Sally A. , Meulia Tea , Hoitink Harry A. J. , Kim Jin-Man
Published: December 2019

Randomly amplified polymorphic DNA (RAPD) analysis and the PCR assay were used in combination with dilution plating on a semiselective medium to detect and enumerate propagules of
Trichoderma hamatum
382, a biocontrol agent utilized in compost-amended mixes. Distinct and reproducible fingerprints were obtained upon amplification of purified genomic DNA of
T. hamatum
382 with the random primers OPE-16, OPH-19, and OPH-20. Three amplified DNA fragments of 0.35 (OPE-16
0.35
), 0.6 (OPH-19
0.6
), and 0.65 (OPH-20
0.65
) kb were diagnostic for
T. hamatum
382, clearly distinguishing it from 53 isolates of four other
Trichoderma
spp. tested. Some isolates of
T. hamatum
shared these low-molecular-weight fragments with
T. hamatum
382. However, RAPD analysis of isolates of
T. hamatum
with all three random primers used in consecutive PCR tests distinguished
T. hamatum
382 from other isolates of
T. hamatum
. These three RAPD amplicons were cloned and sequenced, and pairs of oligonucleotide primers for each cloned fragment were designed. Use of the primers in the PCR assay resulted in the amplification of DNA fragments of the same size as the cloned RAPD fragments from genomic DNA of
T. hamatum
382. A combination of dilution plating on a semiselective medium for
Trichoderma
spp. and PCR, with the RAPD primers OPH-19, OPE-16, and OPH-20 or the three sequence-characterized primers, was used successfully to verify the presence of
T. hamatum
382 propagules in nine different soil, compost, and potting mix samples. All 23
Trichoderma
isolates recovered on semiselective medium from commercial potting mixes fortified with
T. hamatum
382 were identified as
T. hamatum
382, whereas 274
Trichoderma
isolates recovered from the other nine samples were negative in the PCR assay. Thus, this highly specific combination of techniques allowed detection and enumeration of propagules of
T. hamatum
382 in fortified compost-amended potting mixes. Sequence-characterized amplified region markers also facilitated the development of a very simple procedure to amplify DNA of
T. hamatum
382 directly from fortified compost-amended potting mixes.
Cloning, Sequencing, and Characterization of Genomic Subtracted Sequences from
Listeria monocytogenes
Authors: Wu Fone-Mao , Muriana Peter M.
Published: December 2019

Individual sequences of a genomic subtracted, PCR-amplified, mixed-sequence probe (GS probe) were cloned and sequenced. The GS probe differentiated restriction fragment length polymorphism patterns for
Listeria monocytogenes
but did not hybridize with members of other bacterial genera. Sequence analysis identified several
L. monocytogenes
sequences already present in the GenBank database; the putative identities of other sequences were inferred from homology data, and still other sequences did not exhibit significant levels of homology with any GenBank sequences.
Polyphasic Study of the Spatial Distribution of Microorganisms in Mexican Pozol, a Fermented Maize Dough, Demonstrates the Need for Cultivation-Independent Methods To Investigate Traditional Fermentations
Authors: Ampe Frédéric , ben Omar Nabil , Moizan Claire , Wacher Carmen , Guyot Jean-Pierre
Published: December 2019

The distribution of microorganisms in pozol balls, a fermented maize dough, was investigated by a polyphasic approach in which we used both culture-dependent and culture-independent methods, including microbial enumeration, fermentation product analysis, quantification of microbial taxa with 16S rRNA-targeted oligonucleotide probes, determination of microbial fingerprints by denaturing gradient gel electrophoresis (DGGE), and 16S ribosomal DNA gene sequencing. Our results demonstrate that DGGE fingerprinting and rRNA quantification should allow workers to precisely and rapidly characterize the microbial assemblage in a spontaneous lactic acid fermented food. Lactic acid bacteria (LAB) accounted for 90 to 97% of the total active microflora; no streptococci were isolated, although members of the genus
Streptococcus
accounted for 25 to 50% of the microflora.
Lactobacillus plantarum
and
Lactobacillus fermentum
, together with members of the genera
Leuconostoc
and
Weissella
, were the other dominant organisms. The overall activity was more important at the periphery of a ball, where eucaryotes, enterobacteria, and bacterial exopolysacharide producers developed. Our results also showed that the metabolism of heterofermentative LAB was influenced in situ by the distribution of the LAB in the pozol ball, whereas homolactic fermentation was controlled primarily by sugar limitation. We propose that starch is first degraded by amylases from LAB and that the resulting sugars, together with the lactate produced, allow a secondary flora to develop in the presence of oxygen. Our results strongly suggest that cultivation-independent methods should be used to study traditional fermented foods.
Escherichia coli
Resistance to Chlorine and Glutathione Synthesis in Response to Oxygenation and Starvation
Authors: Saby Sébastien , Leroy Pierre , Block Jean-Claude
Published: December 2019

Reduced glutathione (GSH) levels and resistance to chlorine were measured for two isogenic
Escherichia coli
strains stressed by oxygenation and/or starvation. The
E. coli
mutant deficient in GSH was not more sensitive to the oxidant than its parent strain when the bacteria were cultured with a low oxygenation rate. Starvation or oxygenation increased the resistance of the parent strain to chlorine, while the resistance of the deficient strain remained unchanged.
Comparative Survival of Free Shiga Toxin 2-Encoding Phages and
Escherichia coli
Strains outside the Gut
Authors: Muniesa Maite , Lucena Francisco , Jofre Juan
Published: December 2019

The behavior outside the gut of seeded
Escherichia coli
O157:H7, naturally occurring
E. coli
, somatic coliphages, bacteriophages infecting O157:H7, and Shiga toxin 2 (Stx2)-encoding bacteriophages was studied to determine whether the last persist in the environment more successfully than their host bacteria. The ratios between the numbers of
E. coli
and those of the different bacteriophages were clearly lower in river water than in sewage of the area, whereas the ratios between the numbers of the different phages were similar. In addition, the numbers of bacteria decreased between 2 and 3 log units in in situ survival experiments performed in river water, whereas the numbers of phages decreased between 1 and 2 log units. Chlorination and pasteurization treatments that reduced by approximately 4 log units the numbers of bacteria reduced by less than 1 log unit the numbers of bacteriophages. Thus, it can be concluded that Stx2-encoding phages persist longer than their host bacteria in the water environment and are more resistant than their host bacteria to chlorination and heat treatment.
Molecular Detection of Norwalk-Like Caliciviruses in Sewage
Authors: Lodder W. J. , Vinjé J. , van de Heide R. , de Roda Husman A. M. , Leenen E. J. T. M. , Koopmans M. P. G.
Published: December 2019

In this study, Norwalk-like virus (NLV) RNA was detected by reverse transcriptase PCR (RT-PCR) in sewage water concentrates. Sequence analysis of the RT-PCR products revealed identical sequences in stools of patients and related sewage samples. In 6 of 11 outbreak-unrelated follow-up samples, multiple NLV genotypes were present. Levels as high as 10
7
RNA-containing particles per liter were found. These data show that high loads of NLVs may be present in sewage and warrant further studies addressing the efficacy of NLV removal by sewage water treatment processes.
Characterization of a Bacteriocin-Like Substance Produced by a Vaginal
Lactobacillus salivarius
Strain
Authors: Ocaña Virginia S. , Pesce de Ruiz Holgado Aída A. , Nader-Macías María Elena
Published: December 2019

A novel bacteriocin-like substance produced by vaginal
Lactobacillus salivarius
subsp.
salivarius
CRL 1328 with activity against
Enterococcus faecalis
,
Enterococcus faecium
, and
Neisseria gonorrhoeae
was characterized. The highest level of production of this heat-resistant peptide or protein occurred during the late exponential phase. Its mode of action was shown to be bactericidal.
L. salivarius
subsp.
salivarius
CRL 1328 could be used for the design of a probiotic to prevent urogenital infections.
Toxicity, Binding, and Permeability Analyses of Four
Bacillus thuringiensis
Cry1 δ-Endotoxins Using Brush Border Membrane Vesicles of
Spodoptera exigua
and
Spodoptera frugiperda
Authors: Luo Ke , Banks David , Adang Michael J.
Published: December 2019

The binding and pore formation properties of four
Bacillus thuringiensis
Cry1 toxins were analyzed by using brush border membrane vesicles from
Spodoptera exigua
and
Spodoptera frugiperda
, and the results were compared to the results of toxicity bioassays. Cry1Fa was highly toxic and Cry1Ac was nontoxic to
S. exigua
and
S. frugiperda
larvae, while Cry1Ca was highly toxic to
S. exigua
and weakly toxic to
S. frugiperda
. In contrast, Cry1Bb was active against
S. frugiperda
but only marginally active against
S. exigua
. Bioassays performed with iodinated Cry1Bb, Cry1Fa, and Cry1Ca showed that the effects of iodination on toxin activity were different. The toxicities of I-labeled Cry1Bb and Cry1Fa against
Spodoptera
species were significantly less than the toxicities of the unlabeled toxins, while Cry1Ca retained its insecticidal activity when it was labeled with
125
I. Binding assays showed that iodination prevented Cry1Fa from binding to
Spodoptera
brush border membrane vesicles.
125
I-labeled Cry1Ac, Cry1Bb, and Cry1Ca bound with high-affinities to brush border membrane vesicles from
S. exigua
and
S. frugiperda
. Competition binding experiments performed with heterologous toxins revealed two major binding sites. Cry1Ac and Cry1Fa have a common binding site, and Cry1Bb, Cry1C, and Cry1Fa have a second common binding site. No obvious relationship between dissociation of bound toxins from brush border membrane vesicles and toxicity was detected. Cry1 toxins were also tested for the ability to alter the permeability of membrane vesicles, as measured by a light scattering assay. Cry1 proteins toxic to
Spodoptera
larvae permeabilized brush border membrane vesicles, but the extent of permeabilization did not necessarily correlate with in vivo toxicity.
Anaerobic Benzene Biodegradation Linked to Nitrate Reduction
Authors: Burland Siobhan M. , Edwards Elizabeth A.
Published: December 2019

Benzene oxidation to carbon dioxide linked to nitrate reduction was observed in enrichment cultures developed from soil and groundwater microcosms. Benzene biodegradation occurred concurrently with nitrate reduction at a constant ratio of 10 mol of nitrate consumed per mol of benzene degraded. Benzene biodegradation linked to nitrate reduction was associated with cell growth; however, the yield, 8.8 g (dry weight) of cells per mol of benzene, was less than 15% of the predicted yield for benzene biodegradation linked to nitrate reduction. In experiments performed with [
14
C]benzene, approximately 92 to 95% of the label was recovered in
14
CO
2
, while the remaining 5 to 8% was incorporated into the nonvolatile fraction (presumably biomass), which is consistent with the low measured yield. In benzene-degrading cultures, nitrite accumulated stoichiometrically as nitrate was reduced and then was slowly reduced to nitrogen gas. When nitrate was depleted and only nitrite remained, the rate of benzene degradation decreased to almost zero. Based on electron balances, benzene biodegradation appears to be coupled more tightly to nitrate reduction to nitrite than to further reduction of nitrite to nitrogen gas.
High-Affinity Transport of Choline-
O
-Sulfate and Its Use as a Compatible Solute in
Bacillus subtilis
Authors: Nau-Wagner Gabriele , Boch Jens , Le Good J. Ann , Bremer Erhard
Published: December 2019

We report here that the naturally occurring choline ester choline-
O
-sulfate serves as an effective compatible solute for
Bacillus subtilis
, and we have identified a high-affinity ATP-binding cassette (ABC) transport system responsible for its uptake. The osmoprotective effect of this trimethylammonium compound closely matches that of the potent and widely employed osmoprotectant glycine betaine. Growth experiments with a set of
B. subtilis
strains carrying defined mutations in the glycine betaine uptake systems OpuA, OpuC, and OpuD and in the high-affinity choline transporter OpuB revealed that choline-
O
-sulfate was specifically acquired from the environment via OpuC. Competition experiments demonstrated that choline-
O
-sulfate functioned as an effective competitive inhibitor for OpuC-mediated glycine betaine uptake, with a

K
i

of approximately 4 μM. Uptake studies with [1,2-
dimethyl
-
14
C]choline-
O
-sulfate showed that its transport was stimulated by high osmolality, and kinetic analysis revealed that OpuC has high affinity for choline-
O
-sulfate, with a

K
m

value of 4 ± 1 μM and a maximum rate of transport (
V
max
) of 54 ± 3 nmol/min · mg of protein in cells grown in minimal medium with 0.4 M NaCl. Growth studies utilizing a
B. subtilis
mutant defective in the choline to glycine betaine synthesis pathway and natural abundance
13
C nuclear magnetic resonance spectroscopy of whole-cell extracts from the wild-type strain demonstrated that choline-
O
-sulfate was accumulated in the cytoplasm and was not hydrolyzed to choline by
B. subtilis
. In contrast, the osmoprotective effect of acetylcholine for
B. subtilis
is dependent on its biotransformation into glycine betaine. Choline-
O
-sulfate was not used as the sole carbon, nitrogen, or sulfur source, and our findings thus characterize this choline ester as an effective compatible solute and metabolically inert stress compound for
B. subtilis
. OpuC mediates the efficient transport not only of glycine betaine and choline-
O
-sulfate but also of carnitine, crotonobetaine, and γ-butyrobetaine (R. Kappes and E. Bremer, Microbiology 144:83–90, 1998). Thus, our data underscore its crucial role in the acquisition of a variety of osmoprotectants from the environment by
B. subtilis
.
Inactivation of Toluene 2-Monooxygenase in
Burkholderia cepacia
G4 by Alkynes
Authors: Yeager Chris M. , Bottomley Peter J. , Arp Daniel J. , Hyman Michael R.
Published: December 2019

High concentrations of acetylene (10 to 50% [vol/vol] gas phase) were required to inhibit the growth of
Burkholderia cepacia
G4 on toluene, while 1% (vol/vol) (gas phase) propyne or 1-butyne completely inhibited growth. Low concentrations of longer-chain alkynes (C
5
to C
10
) were also effective inhibitors of toluene-dependent growth, and 2- and 3-alkynes were more potent inhibitors than their 1-alkyne counterparts. Exposure of toluene-grown
B. cepacia
G4 to alkynes resulted in the irreversible loss of toluene- and
o
-cresol-dependent O
2
uptake activities, while acetate- and 3-methylcatechol-dependent O
2
uptake activities were unaffected. Toluene-dependent O
2
uptake decreased upon the addition of 1-butyne in a concentration- and time-dependent manner. The loss of activity followed first-order kinetics, with apparent rate constants ranging from 0.25 min
−1
to 2.45 min
−1
. Increasing concentrations of toluene afforded protection from the inhibitory effects of 1-butyne. Furthermore, oxygen, supplied as H
2
O
2
, was required for inhibition by 1-butyne. These results suggest that alkynes are specific, mechanism-based inactivators of toluene 2-monooxygenase in
B. cepacia
G4, although the simplest alkyne, acetylene, was relatively ineffective compared to longer alkynes. Alkene analogs of acetylene and propyne—ethylene and propylene—were not inactivators of toluene 2-monooxygenase activity in
B. cepacia
G4 but were oxidized to their respective epoxides, with apparent

K
s

and
V
max
values of 39.7 μM and 112.3 nmol min
−1
mg of protein
−1
for ethylene and 32.3 μM and 89.2 nmol min
−1
mg of protein
−1
for propylene.
A Mutation That Improves Soluble Recombinant Hemoglobin Accumulation in
Escherichia coli
in Heme Excess
Authors: Weickert Michael J. , Pagratis Maria , Glascock Christopher B. , Blackmore Richard
Published: December 2019

High-level expression of soluble recombinant human hemoglobin (rHb) in
Escherichia coli
was obtained with several hemoglobin variants. Under identical conditions, two rHbs containing the Presbyterian mutation (Asn-108→Lys) in β-globin accumulated to approximately twofold less soluble globin than rHbs containing the corresponding wild-type β-globin subunit accumulated. The β-globin Providence
(asp)
mutation (Lys-82→Asp) significantly improved soluble rHb accumulation compared to the wild-type β-globin subunit and restored soluble accumulation of rHbs containing the Presbyterian mutation to wild-type levels. The Providence
asp
substitution introduced a negatively charged residue into the normally cationic 2,3-bisphosphoglycerate binding pocket, potentially reducing the electrostatic repulsion in the absence of the polyanion. The average soluble globin accumulation when there was coexpression of di-α-globin and β-Lys-82→Asp-globin (rHb9.1) and heme was present in at least a threefold molar excess was 36% ± 3% of the soluble cell protein in
E. coli
. The average total accumulation (soluble globin plus insoluble globin) was 56% ± 7% of the soluble cell protein. Fermentations yielded 6.0 ± 0.3 g of soluble rHb9.1 per liter 16 h after induction and 6.4 ± 0.2 g/liter 24 h after induction. The average total globin yield was 9.4 g/liter 16 h after induction. High-level accumulation of soluble rHb in
E. coli
depends on culture conditions, the protein sequence, and the molar ratio of the heme cofactor added.
Influence of Carbohydrate Starvation and Arginine on Culturability and Amino Acid Utilization of
Lactococcus lactis
subsp.
lactis
Authors: Stuart Mark R. , Chou Lan Szu , Weimer Bart C.
Published: December 2019

Two strains of
Lactococcus lactis
subsp.
lactis
were used to determine the influence of lactose and arginine on viability and amino acid use during carbohydrate starvation. Lactose provided energy for logarithmic-phase growth, and amino acids such as arginine provided energy after carbohydrate exhaustion. Survival time, cell numbers, and ATP concentrations increased with the addition of arginine to the basal medium. By the onset of lactose exhaustion, the concentrations of glycine-valine and glutamate had decreased by as much as 67% in
L. lactis
ML3, whereas the serine concentration increased by 97% during the same period. When no lactose was added, the concentrations of these amino acids remained constant. Similar trends were observed for
L. lactis
11454. Without lactose or arginine,
L. lactis
ML3 was nonculturable on agar but was viable after 2 days, as measured by fluorescent viability stains and intracellular ATP levels. However,
L. lactis
11454 without lactose or arginine remained culturable for at least 14 days. These data suggest that lactococci become viable but nonculturable in response to carbohydrate depletion. Additionally, these data indicate that amino acids other than arginine facilitate the survival of
L. lactis
during carbohydrate starvation.
Purification and Characterization of a Novel 5-Oxoprolinase (without ATP-Hydrolyzing Activity) from
Alcaligenes faecalis
N-38A
Authors: Nishimura Atsuhisa , Ozaki Yasunori , Oyama Hiroshi , Shin Takashi , Murao Sawao
Published: December 2019

A novel type of 5-oxoprolinase was found in a cell extract of strain N-38A, which was later identified as
Alcaligenes faecalis
. The enzyme in the cell extract was purified to a homogeneous state with a yield of 16.6%. The molecular weight of the purified enzyme was estimated to be 47,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, suggesting that the enzyme is a monomeric protein. The enzyme specifically catalyzed a decyclization of
l
-pyroglutamate without hydrolyzing ATP and also without any requirements for metal ions such as Mg
2+
and K
+
. The optimal pH for the decyclization was 7.4. The reaction was reversible. The equilibrium constant of the reaction,
K
eq
= [
l
-glutamate]/[
l
-pyroglutamate], was evaluated to be approximately 0.035, which indicates that the reaction tends to form
l
-pyroglutamate. The amino-terminal amino acid sequence of the enzyme was H-Glu-Pro-Arg-Leu-Asp-Thr-Ser-Gln-Leu-Tyr-Ala-Asp-Val-His-Phe-. No protein with a similar sequence was found in the DNASIS database. Based on these data, it was strongly suggested that the enzyme described here is a novel type of 5-oxoprolinase.
Role of Microcystins in Poisoning and Food Ingestion Inhibition of
Daphnia galeata
Caused by the Cyanobacterium
Microcystis aeruginosa
Authors: Rohrlack Thomas , Dittmann Elke , Henning Manfred , Börner Thomas , Kohl Johannes-Günter
Published: December 2019

The effects of microcystins on
Daphnia galeata
, a typical filter-feeding grazer in eutrophic lakes, were investigated. To do this, the microcystin-producing wild-type strain
Microcystis aeruginosa
PCC7806 was compared with a mcy
−
PCC7806 mutant, which could not synthesize any variant of microcystin due to mutation of a microcystin synthetase gene. The wild-type strain was found to be poisonous to
D. galeata
, whereas the mcy
−
mutant did not have any lethal effect on the animals. Both variants of PCC7806 were able to reduce the
Daphnia
ingestion rate. Our results suggest that microcystins are the most likely cause of the daphnid poisoning observed when wild-type strain PCC7806 is fed to the animals, but these toxins are not responsible for inhibition of the ingestion process.
Control of Flavor Development in Wine during and after Malolactic Fermentation byOenococcus oeni
Authors: Nielsen Jan Clair , Richelieu Marianne
Published: December 2019

During malolactic fermentation in wine byOenococcus oeni, the degradation of citric acid was delayed compared to the degradation of malic acid. The maximum concentration of diacetyl, an intermediary compound in the citric acid metabolism with a buttery or nutty flavor, coincided with the exhaustion of malic acid in the wine. The maximum concentration of diacetyl obtained during malolactic fermentation was strongly dependent on the oxygen concentration and the redox potential of the wine and, to a lesser extent, on the initial citric acid concentration. The final diacetyl concentration in the wine was also dependent on the concentration of SO2. Diacetyl combines rather strongly with SO2(Kf= 7.2 × 103M−1in 0.1 M malate buffer [pH 3.5] at 30°C). The reaction is exothermic and reversible. If the concentration of SO2decreases during storage of the wine, the diacetyl concentration increases again.
Isolation of a Bacterial Strain Able To Degrade Branched Nonylphenol
Authors: Tanghe Tom , Dhooge Willem , Verstraete Willy
Published: December 2019

Conventional enrichment of microorganisms on branched nonylphenol (NP) as only carbon and energy source yielded mixed cultures able to grow on the organic compound. However, plating yielded no single colonies capable, alone or in combination with other isolates, of degrading the NP in liquid culture. Therefore, a special approach was used, referred to as “serial dilution-plate resuspension,” to reduce culture complexity. In this way, one isolate, TTNP3, tentatively identified as a
Sphingomonas
sp., was found to be able to grow on NP in liquid culture. Remarkably, this isolate was able to be filtered through a 0.45-μm-pore-diameter filter. Moreover, isolate TTNP3 did not form visible colonies on mineral medium with NP, and it formed visible colonies on R
2
A agar only after a prolonged incubation of 1 week. High-performance liquid chromatography and gas chromatography-mass spectroscopy analysis of the culture media indicated that the strain starts the degradation of NP with a fission of the phenol ring and preferably uses the
para
isomer of NP and not the
ortho
isomer. No distinct accumulation of an intermediary product could be observed.
Regulatory Factors Associated with Synthesis of the Osmolyte Glycine Betaine in the Halophilic Methanoarchaeon
Methanohalophilus portucalensis
Authors: Lai Mei-Chin , Yang Daw-Renn , Chuang Ming-Jen
Published: December 2019

The halophilic methanoarchaeon
Methanohalophilus portucalensis
can synthesize de novo and accumulate β-glutamine,
N
ɛ
-acetyl-β-lysine, and glycine betaine (betaine) as compatible solutes (osmolytes) when grown at elevated salt concentrations. Both in vivo and in vitro betaine formation assays in this study confirmed previous nuclear magnetic resonance
13
C-labelling studies showing that the de novo synthesis of betaine proceeded from glycine, sarcosine, and dimethylglycine to form betaine through threefold methylation. Exogenous sarcosine (1 mM) effectively suppressed the intracellular accumulation of betaine, and a higher level of sarcosine accumulation was accompanied by a lower level of betaine synthesis. Exogenous dimethylglycine has an effect similar to that of betaine addition, which increased the intracellular pool of betaine and suppressed the levels of
N
ɛ
-acetyl-β-lysine and β-glutamine. Both in vivo and in vitro betaine formation assays with glycine as the substrate showed only sarcosine and betaine, but no dimethylglycine. Dimethylglycine was detected only when it was added as a substrate in in vitro assays. A high level of potassium (400 mM and above) was necessary for betaine formation in vitro. Interestingly, no methylamines were detected without the addition of KCl. Also, high levels of NaCl and LiCl (800 mM) favored sarcosine accumulation, while a lower level (400 mM) favored betaine synthesis. The above observations indicate that a high sarcosine level suppressed multiple methylation while dimethylglycine was rapidly converted to betaine. Also, high levels of potassium led to greater amounts of betaine, while lower levels of potassium led to greater amounts of sarcosine. This finding suggests that the intracellular levels of both sarcosine and potassium are associated with the regulation of betaine synthesis in
M. portucalensis
.
Suppression of Bacterial Blight by a Bacterial Community Isolated from the Guttation Fluids of Anthuriums
Authors: Fukui R. , Fukui H. , Alvarez A. M.
Published: December 2019

Growth and survival of
Xanthomonas campestris
pv. dieffenbachiae in guttation fluids (xylem sap exuded from leaf margins) of anthuriums were suppressed by several bacterial strains indigenous to leaves of various anthurium cultivars. Inhibition of growth was not observed in filter-sterilized guttation fluids and was restored to original levels only by reintroducing specific mixtures of bacteria into filter-sterilized guttation fluids. The inhibitory effect was related to the species in the bacterial community rather than to the total numbers of bacteria in the guttation fluids. One very effective bacterial community consisted of five species isolated from inhibitory guttation fluids of two susceptible anthurium cultivars. The individual strains in this community had no effect on the pathogen, but the mixture was inhibitory to
X. campestris
pv. dieffenbachiae in guttation fluids. The populations of the individual strains remained near the initial inoculum levels for at least 14 days. The effect of the five inhibitory strains on reducing disease in susceptible anthurium plants was tested by using a bioluminescent strain of
X. campestris
pv. dieffenbachiae to monitor the progression of disease in leaves nondestructively. Invasion of the pathogen through hydathodes at leaf margins was reduced by applying the strain mixture to the leaves. When the strain mixture was applied directly to wounds created on the leaf margins, the pathogen failed to invade through the wounds. This bacterial community has potential for biological control of anthurium blight.
Immobilization Patterns and Dynamics of Acetate-Utilizing Methanogens Immobilized in Sterile Granular Sludge in Upflow Anaerobic Sludge Blanket Reactors
Authors: Schmidt Jens Ejbye , Ahring Birgitte Kjær
Published: December 2019

Sterile granular sludge was inoculated with either
Methanosarcina mazeii
S-6,
Methanosaeta concilii
GP-6, or both species in acetate-fed upflow anaerobic sludge blanket (UASB) reactors to investigate the immobilization patterns and dynamics of aceticlastic methanogens in granular sludge. After several months of reactor operation, the methanogens were immobilized, either separately or together. The fastest immobilization was observed in the reactor containing
M. mazeii
S-6. The highest effluent concentration of acetate was observed in the reactor with only
M. mazeii
S-6 immobilized, while the lowest effluent concentration of acetate was observed in the reactor where both types of methanogens were immobilized together. No changes were observed in the kinetic parameters (

K
s

and μ
max
) of immobilized
M. concilii
GP-6 or
M. mazeii
S-6 compared with suspended cultures, indicating that immobilization does not affect the growth kinetics of these methanogens. An enzyme-linked immunosorbent assay using polyclonal antibodies against either
M. concilii
GP-6 or
M. mazeii
S-6 showed significant variations in the two methanogenic populations in the different reactors. Polyclonal antibodies were further used to study the spatial distribution of the two methanogens.
M. concilii
GP-6 was immobilized only on existing support material without any specific pattern.
M. mazeii
S-6, however, showed a different immobilization pattern: large clumps were formed when the concentration of acetate was high, but where the acetate concentration was low this strain was immobilized on support material as single cells or small clumps. The data clearly show that the two aceticlastic methanogens immobilize differently in UASB systems, depending on the conditions found throughout the UASB reactor.
Physiological Characterization of Viable-but-Nonculturable
Campylobacter jejuni
Cells
Authors: Tholozan J. L. , Cappelier J. M. , Tissier J. P. , Delattre G. , Federighi M.
Published: December 2019

Campylobacter jejuni
is a pathogenic, microaerophilic, gram-negative, mesophilic bacterium. Three strains isolated from humans with enteric campylobacteriosis were able to survive at high population levels (10
7
cells ml
−1
) as viable-but-nonculturable (VBNC) forms in microcosm water. The VBNC forms of the three
C. jejuni
strains were enumerated and characterized by using 5-cyano-2,3-ditolyl tetrazolium chloride–4′,6-diamino-2-phenylindole staining. Cellular volume, adenylate energy charge, internal pH, intracellular potassium concentration, and membrane potential values were determined in stationary-phase cell suspensions after 48 h of culture on Columbia agar and after 1 to 30 days of incubation in microcosm water and compared. A notable increase in cell volume was observed with the VBNC state; the average cell volumes were 1.73 μl mg of protein
−1
for the culturable form and 10.96 μl mg of protein
−1
after 30 days of incubation in microcosm water. Both the internal potassium content and the membrane potential were significantly lower in the VBNC state than in the culturable state. Culturable cells were able to maintain a difference of 0.6 to 0.9 pH unit between the internal and external pH values; with VBNC cells this difference decreased progressively with time of incubation in microcosm water. Measurements of the cellular adenylate nucleotide concentrations revealed that the cells had a low adenylate energy charge (0.66 to 0.26) after 1 day of incubation in microcosm water, and AMP was the only nucleotide detected in the three strains after 30 days of incubation in microcosm water.
Randomly Amplified Polymorphic DNA Analysis of Clinical and Environmental Isolates of
Vibrio vulnificus
and Other
Vibrio
Species
Authors: Warner Jennifer M. , Oliver James D.
Published: December 2019

Vibrio vulnificus
is an estuarine bacterium that is capable of causing a rapidly fatal infection in humans. A randomly amplified polymorphic DNA (RAPD) PCR protocol was developed for use in detecting
V. vulnificus
, as well as other members of the genus
Vibrio
. The resulting RAPD profiles were analyzed by using RFLPScan software. This RAPD method clearly differentiated between members of the genus
Vibrio
and between isolates of
V. vulnificus
. Each
V. vulnificus
strain produced a unique band pattern, indicating that the members of this species are genetically quite heterogeneous. All of the vibrios were found to have amplification products whose sizes were within four common molecular weight ranges, while the
V. vulnificus
strains had an additional two molecular weight range bands in common. All of the
V. vulnificus
strains isolated from clinical specimens produced an additional band that was only occasionally found in environmental strains; this suggests that, as is the case with the Kanagawa hemolysin of
Vibrio parahaemolyticus
, the presence of this band may be correlated with the ability of a strain to produce an infection in humans. In addition, band pattern differences were observed between encapsulated and nonencapsulated isogenic morphotypes of the same strain of
V. vulnificus
.
Identification of a Novel Group of Bacteria in Sludge from a Deteriorated Biological Phosphorus Removal Reactor
Authors: Nielsen Alex T. , Liu Wen-Tso , Filipe Carlos , Grady Leslie , Molin Søren , Stahl David A.
Published: December 2019

The microbial diversity of a deteriorated biological phosphorus removal reactor was investigated by methods not requiring direct cultivation. The reactor was fed with media containing acetate and high levels of phosphate (P/C weight ratio, 8:100) but failed to completely remove phosphate in the effluent and showed very limited biological phosphorus removal activity. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA was used to investigate the bacterial diversity. Up to 11 DGGE bands representing at least 11 different sequence types were observed; DNA from the 6 most dominant of these bands was further isolated and sequenced. Comparative phylogenetic analysis of the partial 16S rRNA sequences suggested that one sequence type was affiliated with the alpha subclass of the
Proteobacteria
, one was associated with the
Legionella
group of the gamma subclass of the
Proteobacteria
, and the remaining four formed a novel group of the gamma subclass of the
Proteobacteria
with no close relationship to any previously described species. The novel group represented approximately 75% of the PCR-amplified DNA, based on the DGGE band intensities. Two oligonucleotide rRNA probes for this novel group were designed and used in a whole-cell hybridization analysis to investigate the abundance of this novel group in situ. The bacteria were coccoid and 3 to 4 μm in diameter and represented approximately 35% of the total population, suggesting a relatively close agreement with the results obtained by the PCR-based DGGE method. Further, based on electron microscopy and standard staining microscopic analysis, this novel group was able to accumulate granule inclusions, possibly consisting of polyhydroxyalkanoate, inside the cells.
In Situ Identification of Cyanobacteria with Horseradish Peroxidase-Labeled, rRNA-Targeted Oligonucleotide Probes
Authors: Schönhuber Wilhelm , Zarda Boris , Eix Stella , Rippka Rosmarie , Herdman Michael , Ludwig Wolfgang , Amann Rudolf
Published: December 2019

Individual cyanobacterial cells are normally identified in environmental samples only on the basis of their pigmentation and morphology. However, these criteria are often insufficient for the differentiation of species. Here, a whole-cell hybridization technique is presented that uses horseradish peroxidase (HRP)-labeled, rRNA-targeted oligonucleotides for in situ identification of cyanobacteria. This indirect method, in which the probe-conferred enzyme has to be visualized in an additional step, was necessary since fluorescently monolabeled oligonucleotides were insufficient to overstain the autofluorescence of the target cells. Initially, a nonfluorescent detection assay was developed and successfully applied to cyanobacterial mats. Later, it was demonstrated that tyramide signal amplification (TSA) resulted in fluorescent signals far above the level of autofluorescence. Furthermore, TSA-based detection of HRP was more sensitive than that based on nonfluorescent substrates. Critical points of the assay, such as cell fixation and permeabilization, specificity, and sensitivity, were systematically investigated by using four oligonucleotides newly designed to target groups of cyanobacteria.
Augmentation of Killing of
Escherichia coli
O157 by Combinations of Lactate, Ethanol, and Low-pH Conditions
Authors: Jordan Sarah L. , Glover Jayne , Malcolm Laura , Thomson-Carter Fiona M. , Booth Ian R. , Park Simon F.
Published: December 2019

The acid tolerance of
Escherichia coli
O157:H7 strains can be overcome by addition of lactate, ethanol, or a combination of the two agents. Killing can be increased by as much as 4 log units in the first 5 min of incubation at pH 3 even for the most acid-tolerant isolates. Exponential-phase, habituated, and stationary-phase cells are all sensitive to incubation with lactate and ethanol. Killing correlates with disruption of the capacity for pH homeostasis. Habituated and stationary-phase cells can partially offset the effects of the lowering of cytoplasmic pH.
Glycine Betaine: Reserve Form of Choline in
Penicillium fellutanum
in Low-Sulfate Medium
Authors: Park Yong-Il , Buszko Marian L. , Gander John E.
Published: December 2019

In spite of choline’s importance in fungal metabolism, its sources in cytoplasm have not been fully established.
13
C nuclear magnetic resonance analysis of mycelial extracts from day-5
Penicillium fellutanum
cultures showed that, as well as choline-
O
-sulfate, intracellular glycine betaine is another reserve form of choline, depending on the availability of sulfate in the culture medium. These observations are discussed relative to the multiple roles of choline and its precursors in
P. fellutanum
.
Role of Surface Proteins in
Vibrio cholerae
Attachment to Chitin
Authors: Tarsi Renato , Pruzzo Carla
Published: December 2019

The role of surface proteins in
Vibrio cholerae
attachment to chitin particles in vitro was studied. Treatment of
V. cholerae
O1 ATCC 14034 and ATCC 14035 with pronase E reduced the attachment of bacteria to chitin particles by 57 to 77%. A statistically significant reduction was also observed when the attachment to chitin was evaluated in the presence of homologous Sarkosyl-insoluble membrane proteins (MPs) (67 to 84%),
N
-acetylglucosamine (GlcNAc) (62%), the sugar that makes up chitin, and wheat germ agglutinin (40 to 56%), a lectin that binds GlcNAc. The soluble oligomers
N
,
N
′-diacetylchitobiose or
N
,
N
′,
N
"-triacetylchitotriose caused an inhibition of 14 to 23%. Sarkosyl-insoluble MPs able to bind chitin particles were isolated and visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; two of these peptides (molecular sizes, 36 and 53 kDa) specifically bind GlcNAc.
A Search for Ligninolytic Peroxidases in the Fungus
Pleurotus eryngii
Involving α-Keto-γ-Thiomethylbutyric Acid and Lignin Model Dimers
Authors: Caramelo Lucília , Martínez María Jesús , Martínez Ángel T.
Published: December 2019

Because there is some controversy concerning the ligninolytic enzymes produced by
Pleurotus
species, ethylene release from α-keto-γ-thiomethylbutyric acid (KTBA), as described previously for
Phanerochaete chrysosporium
lignin peroxidase (LiP), was used to assess the oxidative power of
Pleurotus eryngii
cultures and extracellular proteins. Lignin model dimers were used to confirm the ligninolytic capabilities of enzymes isolated from liquid and solid-state fermentation (SSF) cultures. Three proteins that oxidized KTBA in the presence of veratryl alcohol and H
2
O
2
were identified (two proteins were found in liquid cultures, and one protein was found in SSF cultures). These proteins are versatile peroxidases that act on Mn
2+
, as well as on simple phenols and veratryl alcohol. The two peroxidases obtained from the liquid culture were able to degrade a nonphenolic β-O-4 dimer, yielding veratraldehyde, as well as a phenolic dimer which is not efficiently oxidized by
P. chrysosporium
peroxidases. The former reaction is characteristic of LiP. The third KTBA-oxidizing peroxidase oxidized only the phenolic dimer (in the presence of Mn
2+
). Finally, a fourth Mn
2+
-oxidizing peroxidase was identified in the SSF cultures on the basis of its ability to oxidize KTBA in the presence of Mn
2+
. This enzyme is related to the Mn-dependent peroxidase of
P. chrysosporium
because it did not exhibit activity with veratryl alcohol and Mn-independent activity with dimers. These results show that
P. eryngii
produces three types of peroxidases that have the ability to oxidize lignin but lacks a typical LiP. Similar enzymes (in terms of N-terminal sequence and catalytic properties) are produced by other
Pleurotus
species. Some structural aspects of
P. eryngii
peroxidases related to the catalytic properties are discussed.
Bacterivory Rate Estimates and Fraction of Active Bacterivores in Natural Protist Assemblages from Aquatic Systems
Authors: González Juan M.
Published: December 2019

Unlike the fraction of active bacterioplankton, the fraction of active bacterivores (i.e., those involved in grazing) during a specified time period has not been studied yet. Fractions of protists actively involved in bacterivory were estimated assuming that the distributions of bacteria and fluorescently labeled bacteria (FLB) ingested by protists follow Poisson distributions. Estimates were compared with experimental data obtained from FLB uptake experiments. The percentages of protists with ingested FLB (experimental) and the estimates obtained from Poisson distributions were similar for both flagellates and ciliates. Thus, the fraction of protists actively grazing on natural bacteria during a given time period could be estimated. The fraction of protists with ingested bacteria depends on the incubation time and reaches a saturating value. Aquatic systems with very different characteristics were analyzed; estimates of the fraction of protists actively grazing on bacteria ranged from 7 to 100% in the studied samples. Some nanoflagellates appeared to be grazing on specific bacterial sizes. Evidence indicated that there was no discrimination for or against bacterial surrogates (i.e., FLB); also, bacteria were randomly encountered by bacterivorous protists during these short-term uptake experiments. These analyses made it possible to estimate the ingestion rates from FLB uptake experiments by counting the number of flagellates containing ingested FLB. These results represent the first reported estimates of active bacterivores in natural aquatic systems; also, a proposed protocol for estimating in situ ingestion rates by protists represents a significant improvement and simplification to the current protocol and avoids the tedious work of counting the number of ingested FLB per protist.
Reduction of Cell Lysate Viscosity during Processing of Poly(3-Hydroxyalkanoates) by Chromosomal Integration of the Staphylococcal Nuclease Gene in
Pseudomonas putida
Authors: Boynton Zhuang L. , Koon Joseph J. , Brennan Elaine M. , Clouart Jeralyn D. , Horowitz Daniel M. , Gerngross Tillman U. , Huisman Gjalt W.
Published: December 2019

Poly(3-hydroxyalkanoates) (PHAs) are biodegradable thermoplastics which are accumulated by many bacterial species in the form of intracellular granules and which are thought to serve as reserves of carbon and energy.
Pseudomonas putida
accumulates a polyester, composed of medium-side-chain 3-hydroxyalkanoic acids, which has excellent film-forming properties. Industrial processing of PHA involves purification of the PHA granules from high-cell-density cultures. After the fermentation process, cells are lysed by homogenization and PHA granules are purified by chemical treatment and repeated washings to yield a PHA latex. Unfortunately, the liberation of chromosomal DNA during lysis causes a dramatic increase in viscosity, which is problematic in the subsequent purification steps. Reduction of the viscosity is generally achieved by the supplementation of commercially available nuclease preparations or by heat treatment; however, both procedures add substantial costs to the process. As a solution to this problem, a nuclease-encoding gene from
Staphylococcus aureus
was integrated into the genomes of several PHA producers. Staphylococcal nuclease is readily expressed in PHA-producing
Pseudomonas
strains and is directed to the periplasm, and occasionally to the culture medium, without affecting PHA production or strain stability. During downstream processing, the viscosity of the lysate from a nuclease-integrated
Pseudomonas
strain was reduced to a level similar to that observed for the wild-type strain after treatment with commercial nuclease. The nuclease gene was also functionally integrated into the chromosomes of other PHA producers, including
Ralstonia eutropha
.
Microbiological and Geochemical Characterization of Fluvially Deposited Sulfidic Mine Tailings
Authors: Wielinga Bruce , Lucy Juliette K. , Moore Johnnie N. , Seastone October F. , Gannon James E.
Published: December 2019

The fluvial deposition of mine tailings generated from historic mining operations near Butte, Montana, has resulted in substantial surface and shallow groundwater contamination along Silver Bow Creek. Biogeochemical processes in the sediment and underlying hyporheic zone were studied in an attempt to characterize interactions consequential to heavy-metal contamination of shallow groundwater. Sediment cores were extracted and fractionated based on sediment stratification. Subsamples of each fraction were assayed for culturable heterotrophic microbiota, specific microbial guilds involved in metal redox transformations, and both aqueous- and solid-phase geochemistry. Populations of cultivable Fe(III)-reducing bacteria were most prominent in the anoxic, circumneutral pH regions associated with a ferricrete layer or in an oxic zone high in organic carbon and soluble iron. Sulfur- and iron-oxidizing bacteria were distributed in discrete zones throughout the tailings and were often recovered from sections at and below the anoxic groundwater interface. Sulfate-reducing bacteria were also widely distributed in the cores and often occurred in zones overlapping iron and sulfur oxidizers. Sulfate-reducing bacteria were consistently recovered from oxic zones that contained high concentrations of metals in the oxidizable fraction. Altogether, these results suggest a highly varied and complex microbial ecology within a very heterogeneous geochemical environment. Such physical and biological heterogeneity has often been overlooked when remediation strategies for metal contaminated environments are formulated.