bluuebunny/crossref_metadata_2025 huggingface dataset:
Pathogenicity of
Vibrio alginolyticus
for Cultured Gilt-Head Sea Bream (
Sparus aurata
L.)
Authors: Balebona M. Carmen , Andreu Manuel J. , Bordas M. Angeles , Zorrilla Irene , Moriñigo Miguel A. , Borrego Juan J.
Published: December 2019

The in vivo and in vitro pathogenic activities of whole cells and extracellular products of
Vibrio alginolyticus
for cultured gilt-head sea bream were evaluated. The 50% lethal doses ranged from 5.4 × 10
4
to 1.0 × 10
6
CFU/g of body weight. The strains examined had the ability to adhere to skin, gill, and intestinal mucus of sea bream and to cultured cells of a chinook salmon embryo cell line. In addition, the in vitro ability of
V. alginolyticus
to adhere to mucus and skin cells of sea bream was demonstrated by scanning electron microscopy. The biological activities of extracellular products of
V. alginolyticus
were hydrolytic activities; the products were able to degrade sea bream mucus.
V. alginolyticus
was cytotoxic for fish cell lines and lethal for sea bream. Moreover, the extracellular products could degrade sea bream tissues. However, experiments performed with the bath immersion inoculation technique demonstrated that
V. alginolyticus
should be considered a pathogen for sea bream only when the mucus layer is removed and the skin is damaged.
Comparison of the Thermostability Properties of Three Acid Phosphatases from Molds:
Aspergillus fumigatus
Phytase,
A. niger
Phytase, and
A. niger
pH 2.5 Acid Phosphatase
Authors: Wyss Markus , Pasamontes Luis , Rémy Roland , Kohler Josiane , Kusznir Eric , Gadient Martin , Müller Francis , van Loon Adolphus P. G. M.
Published: December 2019

Enzymes that are used as animal feed supplements should be able to withstand temperatures of 60 to 90°C, which may be reached during the feed pelleting process. The thermostability properties of three histidine acid phosphatases,
Aspergillus fumigatus
phytase,
Aspergillus niger
phytase, and
A. niger
optimum pH 2.5 acid phosphatase, were investigated by measuring circular dichroism, fluorescence, and enzymatic activity. The phytases of
A. fumigatus
and
A. niger
were both denatured at temperatures between 50 and 70°C. After heat denaturation at temperatures up to 90°C,
A. fumigatus
phytase refolded completely into a nativelike, fully active conformation, while in the case of
A. niger
phytase exposure to 55 to 90°C was associated with an irreversible conformational change and with losses in enzymatic activity of 70 to 80%. In contrast to these two phytases,
A. niger
pH 2.5 acid phosphatase displayed considerably higher thermostability; denaturation, conformational changes, and irreversible inactivation were observed only at temperatures of ≥80°C. In feed pelleting experiments performed at 75°C, the recoveries of the enzymatic activities of the three acid phosphatases were similar (63 to 73%). At 85°C, however, the recovery of enzymatic activity was considerably higher for
A. fumigatus
phytase (51%) than for
A. niger
phytase (31%) or pH 2.5 acid phosphatase (14%). These findings confirm that
A. niger
pH 2.5 acid phosphatase is irreversibly inactivated at temperatures above 80°C and that the capacity of
A. fumigatus
phytase to refold properly after heat denaturation may favorably affect its pelleting stability.
Degradation of Phthalate and Di-(2-Ethylhexyl)phthalate by Indigenous and Inoculated Microorganisms in Sludge-Amended Soil
Authors: Roslev Peter , Madsen Peter L. , Thyme Jesper B. , Henriksen Kaj
Published: December 2019

The metabolism of phthalic acid (PA) and di-(2-ethylhexyl)phthalate (DEHP) in sludge-amended agricultural soil was studied with radiotracer techniques. The initial rates of metabolism of PA and DEHP (4.1 nmol/g [dry weight]) were estimated to be 731.8 and 25.6 pmol/g (dry weight) per day, respectively. Indigenous microorganisms assimilated 28 and 17% of the carbon in [
14
C]PA and [
14
C]DEHP, respectively, into microbial biomass. The rates of DEHP metabolism were much greater in sludge assays without soil than in assays with sludge-amended soil. Mineralization of [
14
C]DEHP to
14
CO
2
increased fourfold after inoculation of sludge and soil samples with DEHP-degrading strain SDE 2. The elevated mineralization potential was maintained for more than 27 days. Experiments performed with strain SDE 2 suggested that the bioavailability and mineralization of DEHP decreased substantially in the presence of soil and sludge components. The microorganisms metabolizing PA and DEHP in sludge and sludge-amended soil were characterized by substrate-specific radiolabelling, followed by analysis of
14
C-labelled phospholipid ester-linked fatty acids (
14
C-PLFAs). This assay provided a radioactive fingerprint of the organisms actively metabolizing [
14
C]PA and [
14
C]DEHP. The
14
C-PLFA fingerprints showed that organisms with different PLFA compositions metabolized PA and DEHP in sludge-amended soil. In contrast, microorganisms with comparable
14
C-PLFA fingerprints were found to dominate DEHP metabolism in sludge and sludge-amended soil. Our results suggested that indigenous sludge microorganisms dominated DEHP degradation in sludge-amended soil. Mineralization of DEHP and PA followed complex kinetics that could not be described by simple first-order equations. The initial mineralization activity was described by an exponential function; this was followed by a second phase that was described best by a fractional power function. In the initial phase, the half times for PA and DEHP in sludge-amended soil were 2 and 58 days, respectively. In the late phase of incubation, the apparent half times for PA and DEHP increased to 15 and 147 days, respectively. In the second phase (after more than 28 days), the half time for DEHP was 2.9 times longer in sludge-amended soil assays than in sludge assays without soil. Experiments with radiolabelled DEHP degraders suggested that a significant fraction of the
14
CO
2
produced in long-term degradation assays may have originated from turnover of labelled microbial biomass rather than mineralization of [
14
C]PA or [
14
C]DEHP. It was estimated that a significant amount of DEHP with poor biodegradability and extractability remains in sludge-amended soil for extended periods of time despite the presence of microorganisms capable of degrading the compound (e.g., more than 40% of the DEHP added is not mineralized after 1 year).
Purification, Characterization, and Molecular Analysis of Thermostable Cellulases CelA and CelB from
Thermotoga neapolitana
Authors: Bok Jin-Duck , Yernool Dinesh A. , Eveleigh Douglas E.
Published: December 2019

Two thermostable endocellulases, CelA and CelB, were purified from
Thermotoga neapolitana
. CelA (molecular mass, 29 kDa; pI 4.6) is optimally active at pH 6.0 at 95°C, while CelB (molecular mass, 30 kDa; pI 4.1) has a broader optimal pH range (pH 6.0 to 6.6) at 106°C. Both enzymes are characterized by a high level of activity (high
V
max
value and low apparent

K
m

value) with carboxymethyl cellulose; the specific activities of CelA and CelB are 1,219 and 1,536 U/mg, respectively. With
p
-nitrophenyl cellobioside the
V
max
values of CelA and CelB are 69.2 and 18.4 U/mg, respectively, while the

K
m

values are 0.97 and 0.3 mM, respectively. The major end products of cellulose hydrolysis, glucose and cellobiose, competitively inhibit CelA, and CelB. The

K
i

values for CelA are 0.44 M for glucose and 2.5 mM for cellobiose; the

K
i

values for CelB are 0.2 M for glucose and 1.16 mM for cellobiose. CelB preferentially cleaves larger cellooligomers, producing cellobiose as the end product; it also exhibits significant transglycosylation activity. This enzyme is highly thermostable and has half-lives of 130 min at 106°C and 26 min at 110°C. A single clone encoding the
celA
and
celB
genes was identified by screening a
T. neapolitana
genomic library in
Escherichia coli
. The
celA
gene encodes a 257-amino-acid protein, while
celB
encodes a 274-amino-acid protein. Both proteins belong to family 12 of the glycosyl hydrolases, and the two proteins are 60% similar to each other. Northern blots of
T. neapolitana
mRNA revealed that
celA
and
celB
are monocistronic messages, and both genes are inducible by cellobiose and are repressed by glucose.
Isolation, Purification, and Characterization of the PR Oxidase from
Penicillium roqueforti
Authors: Chang Shenq-Chyi , Lei Wen-Yee , Tsai Ying-Chieh , Wei Yau-Huei
Published: December 2019

The PR oxidase, an extracellular enzyme, involved in the conversion of PR toxin into PR acid, was purified from the culture broth of
Penicillium roqueforti
ATCC 48936. The enzyme has a pI of 4.5 and a molecular mass of approximately 88 kDa, and it is a monomer. The optimum pH for this enzyme is ca. 4.0, and the optimum temperature is 50°C.
Detection of
Mycoplasma hyopneumoniae
by Air Sampling with a Nested PCR Assay
Authors: Stärk Katharina D. C. , Nicolet Jacques , Frey Joachim
Published: December 2019

This article describes the first successful detection of airborne
Mycoplasma hyopneumoniae
under experimental and field conditions with a new nested PCR assay. Air was sampled with polyethersulfone membranes (pore size, 0.2 μm) mounted in filter holders. Filters were processed by dissolution and direct extraction of DNA for PCR analysis. For the PCR, two nested pairs of oligonucleotide primers were designed by using an
M. hyopneumoniae
-specific DNA sequence of a repeated gene segment. A nested PCR assay was developed and used to analyze samples collected in eight pig houses where respiratory problems had been common. Air was also sampled from a mycoplasma-free herd. The nested PCR was highly specific and 10
4
times as sensitive as a one-step PCR. Under field conditions, the sampling system was able to detect airborne
M. hyopneumoniae
on 80% of farms where acute respiratory disease was present. No airborne
M. hyopneumoniae
was detected on infected farms without acute cases. The chance of successful detection was increased if air was sampled at several locations within a room and at a lower air humidity.
Photometric Application of the Gram Stain Method To Characterize Natural Bacterial Populations in Aquatic Environments
Authors: Saida H. , Ytow N. , Seki H.
Published: December 2019

The Gram stain method was applied to the photometric characterization of aquatic bacterial populations with a charge-coupled device camera and an image analyzer.Escherichia coliandBacillus subtiliswere used as standards of typical gram-negative and gram-positive bacteria, respectively. A mounting agent to obtain clear images of Gram-stained bacteria on Nuclepore membrane filters was developed. The bacterial stainability by the Gram stain was indicated by the Gram stain index (GSI), which was applicable not only to the dichotomous classification of bacteria but also to the characterization of cell wall structure. The GSI spectra of natural bacterial populations in water with various levels of eutrophication showed a distinct profile, suggesting possible staining specificity that indicates the presence of a particular bacterial population in the aquatic environment.
Characterization of the Cricket Hindgut Microbiota with Fluorescently Labeled rRNA-Targeted Oligonucleotide Probes
Authors: Santo Domingo Jorge W. , Kaufman Michael G. , Klug Michael J. , Tiedje James M.
Published: December 2019

Most cricket hindgut microorganisms (60 to 80%) were detected with a universal fluorescent rRNA-targeted probe and found to be eubacteria. Group-specific probes showed that the hindguts of five different cricket species harbor similar bacterial groups, although in different proportions, and that different diets shifted the structure of the hindgut microbial community. The
Bacteroides-Prevotella
probe, of the eight eubacterial probes tested, stained the largest percentage of cells in all crickets.
Detection of mRNA by Reverse Transcription-PCR as an Indicator of Viability in
Escherichia coli
Cells
Authors: Sheridan G. E. C. , Masters C. I. , Shallcross J. A. , Mackey B. M.
Published: December 2019

The relationship between the detection of mRNA and cellular viability in
Escherichia coli
was investigated in cells killed by heat or ethanol. Reverse transcription-PCR (RT-PCR) methods were developed for detecting mRNA from
rpoH
,
groEL
, and
tufA
genes. mRNA from all three genes was detected immediately after the cells had been killed by heat or ethanol but gradually disappeared with time when dead cells were held at room temperature. In heat-killed cells, some mRNA targets became undetectable after 2 to 16 h, whereas after ethanol treatment, mRNA was still detected after 16 h. In contrast, 16S rRNA was detected by RT-PCR in all samples containing dead cells and did not disappear during a subsequent incubation of 16 h at room temperature. Of the different types of nucleic acid, mRNA is the most promising candidate for an indicator of viability in bacteria, but its persistence in dead cells depends on the inactivating treatment and subsequent holding conditions.
Effect of Temperature on Adhesion of
Vibrio
Strain AK-1 to
Oculina patagonica
and on Coral Bleaching
Authors: Toren A. , Landau L. , Kushmaro A. , Loya Y. , Rosenberg E.
Published: December 2019

Laboratory aquarium experiments demonstrated that
Vibrio
strain AK-1 caused rapid and extensive bleaching of the coral
Oculina patagonica
at 29°C, slower and less-complete bleaching at 23°C, and no bleaching at 16°C. At 29°C, the application of approximately 100
Vibrio
strain AK-1 cells directly onto the coral caused 50 and 83% bleaching after 10 and 20 days, respectively. At 16°C, there was no bleaching, even with an initial inoculum of 1.2 × 10
8
bacteria. To begin to understand the effect of seawater temperature on bleaching of
O. patagonica
by
Vibrio
strain AK-1, adhesion of the bacteria to the coral as a function of temperature was studied. Inoculation of 10
7
Vibrio
strain AK-1 organisms into flasks containing 20 ml of seawater at 25°C and a fragment of
O. patagonica
resulted in net levels of bacterial adhesion to the coral of 45, 78, and 84% after 2, 6, and 8 h, respectively. The adhesion was inhibited 65% by 0.001%
d
-galactose and 94% by 0.001% methyl-β-
d
-galactopyranoside (β-M-Gal). After the incubation of
Vibrio
strain AK-1 with the coral for 6 h, 42% of the input bacteria were released from the coral with 0.01% β-M-Gal, compared to less than 0.2% when β-M-Gal was present during the adhesion step. Adhesion did not occur when
Vibrio
strain AK-1 was grown at 16°C, regardless of whether the corals were maintained at 16 or 25°C, whereas bacteria grown at 25°C adhered to corals maintained at 16 or 25°C. Bacteria grown at 25°C adhered avidly to Sepharose beads containing covalently bound β-
d
-galactopyranoside but failed to bind if grown at 16°C. These data suggest that elevated seawater temperatures may cause coral bleaching by allowing for the expression of adhesin genes of
Vibrio
strain AK-1.
Transformation of
Acinetobacter
sp. Strain BD413 by Transgenic Sugar Beet DNA
Authors: Gebhard Frank , Smalla Kornelia
Published: December 2019

The ability of
Acinetobacter
sp. strain BD413(pFG4Δ
npt
II) to take up and integrate transgenic plant DNA based on homologous recombination was studied under optimized laboratory conditions. Restoration of
npt
II, resulting in kanamycin-resistant transformants, was observed with plasmid DNA, plant DNA, and homogenates carrying the gene
npt
II. Molecular analysis showed that some transformants not only restored the 317-bp deletion but also obtained additional DNA.
Biodegradation of Aliphatic-Aromatic Copolyesters by
Thermomonospora fusca
and Other Thermophilic Compost Isolates
Authors: Kleeberg Ilona , Hetz Claudia , Kroppenstedt Reiner Michael , Müller Rolf-Joachim , Deckwer Wolf-Dieter
Published: December 2019

Random aliphatic-aromatic copolyesters synthesized from 1,4-butanediol, adipic acid, and terephthalic acid (BTA) have excellent thermal and mechanical properties and are biodegradable by mixed cultures (e.g., in compost). Over 20 BTA-degrading strains were isolated by using compost as a microbial source. Among these microorganisms, thermophilic actinomycetes obviously play an outstanding role and appear to dominate the initial degradation step. Two actinomycete strains exhibited about 20-fold higher BTA degradation rates than usually observed in a common compost test. These isolates were identified as
Thermomonospora fusca
strains. They appeared to be particularly suitable for establishment of rapid degradation tests and were used in comparative studies on the biodegradation of various polyesters.
Transcriptional Activation of the
Aspergillus nidulans gpdA
Promoter by Osmotic Signals
Authors: Redkar Rajendra J. , Herzog Roland W. , Singh Narendra K.
Published: December 2019

A differentially expressed
gpdA
cDNA clone was isolated from NaCl-adapted
Aspergillus nidulans
(FGSC359) and identified as glyceraldehyde-3-phosphate dehydrogenase (
gpdA
) on the basis of its nucleotide sequence. The level of
gpdA
RNA substantially increased in cultures gradually adapted to NaCl but was greatly reduced in cultures exposed briefly to a high concentration of NaCl. A
pyrG
auxotroph of
A. nidulans
(A773) was cotransformed with a
gpdA-uidA
construct and a plasmid containing the
Neurospora crassa pyr4
gene as a selectable marker. One
pyrG
+
β-glucuronidase-positive (GUS
+
) transformant was selected, and stable integration of the
gpdA-uidA
construct into the genome was confirmed by Southern blot analysis. Gradual adaptation to increasing concentrations of NaCl resulted in an increase in GUS activity to 2.7-fold. GUS activity was reduced after a 2-h exposure of an unadapted culture to 2 M NaCl but gradually increased to a maximum of twofold after 24 h. GUS activity also increased by 8.4-fold in Na
2
SO
4
-adapted cultures, 4.9-fold in polyethylene glycol-adapted cultures, and 7.5-fold in KCl-adapted cultures. These results are consistent with the hypothesis that the
A. nidulans gpdA
promoter is transcriptionally activated by osmotic signals.
Growth of
Geobacter sulfurreducens
with Acetate in Syntrophic Cooperation with Hydrogen-Oxidizing Anaerobic Partners
Authors: Cord-Ruwisch Ralf , Lovley Derek R. , Schink Bernhard
Published: December 2019

Pure cultures of
Geobacter sulfurreducens
and other Fe(III)-reducing bacteria accumulated hydrogen to partial pressures of 5 to 70 Pa with acetate, butyrate, benzoate, ethanol, lactate, or glucose as the electron donor if electron release to an acceptor was limiting.
G. sulfurreducens
coupled acetate oxidation with electron transfer to an anaerobic partner bacterium in the absence of ferric iron or other electron acceptors. Cocultures of
G. sulfurreducens
and
Wolinella succinogenes
with nitrate as the electron acceptor degraded acetate efficiently and grew with doubling times of 6 to 8 h. The hydrogen partial pressures in these acetate-degrading cocultures were considerably lower, in the range of 0.02 to 0.04 Pa. From these values and the concentrations of the other reactants, it was calculated that in this cooperation the free energy change available to
G. sulfurreducens
should be about −53 kJ per mol of acetate oxidized, assuming complete conversion of acetate to CO
2
and H
2
. However, growth yields (18.5 g of dry mass per mol of acetate for the coculture, about 14 g for
G. sulfurreducens
) indicated considerably higher energy gains. These yield data, measurement of hydrogen production rates, and calculation of the diffusive hydrogen flux indicated that electron transfer in these cocultures may not proceed exclusively via interspecies hydrogen transfer but may also proceed through an alternative carrier system with higher redox potential, e.g., a
c
-type cytochrome that was found to be excreted by
G. sulfurreducens
into the culture fluid. Syntrophic acetate degradation was also possible with
G. sulfurreducens
and
Desulfovibrio desulfuricans
CSN but only with nitrate as electron acceptor. These cultures produced cell yields of 4.5 g of dry mass per mol of acetate, to which both partners contributed at about equal rates. These results demonstrate that some Fe(III)-reducing bacteria can oxidize organic compounds under Fe(III) limitation with the production of hydrogen, and they provide the first example of rapid acetate oxidation via interspecies electron transfer at moderate temperature.
Mycobacterium aviumBacilli Grow Saprozoically in Coculture withAcanthamoeba polyphagaand Survive within Cyst Walls
Authors: Steinert Michael , Birkness Kristin , White Elizabeth , Fields Barry , Quinn Frederick
Published: December 2019

Protozoans are gaining recognition as environmental hosts for a variety of waterborne pathogens. We compared the growth ofMycobacterium avium, a human pathogen associated with domestic water supplies, in coculture with the free-living amoebaAcanthamoeba polyphagawith the growth ofM. aviumwhen it was separated from amoebae by a 0.1-μm-pore-size polycarbonate membrane (in a parachamber). Although viable mycobacteria were observed within amoebal vacuoles, there was no significant difference between bacterial growth in coculture and bacterial growth in the parachamber. This suggests thatM. aviumis able to grow saprozoically on products secreted by the amoebae. In contrast,Legionella pneumophila, a well-studied intracellular parasite of amoebae, multiplied only in coculture. A comparison of amoebae infected withL. pneumophilaand amoebae infected withM. aviumby electron microscopy demonstrated that there were striking differences in the locations of the bacteria within amoebal cysts. WhileL. pneumophilaresided within the cysts,M. aviumwas found within the outer walls of the double-walled cysts ofA. polyphaga. These locations may provide a reservoir for the bacteria when environmental conditions become unfavorable.
Comparison of Membrane Filtration and Multiple-Tube Fermentation by the Colilert and Enterolert Methods for Detection of Waterborne Coliform Bacteria,
Escherichia coli
, and Enterococci Used in Drinking and Bathing Water Quality Monitoring in Southern Sweden
Authors: Eckner Karl F.
Published: December 2019

A total of 338 water samples, 261 drinking water samples and 77 bathing water samples, obtained for routine testing were analyzed in duplicate by Swedish standard methods using multiple-tube fermentation or membrane filtration and by the Colilert and/or Enterolert methods. Water samples came from a wide variety of sources in southern Sweden (Skåne). The Colilert method was found to be more sensitive than Swedish standard methods for detecting coliform bacteria and of equal sensitivity for detecting
Escherichia coli
when all drinking water samples were grouped together. Based on these results, Swedac, the Swedish laboratory accreditation body, approved for the first time in Sweden use of the Colilert method at this laboratory for the analysis of all water sources not falling under public water regulations (A-krav). The coliform detection study of bathing water yielded anomalous results due to confirmation difficulties.
E. coli
detection in bathing water was similar by both the Colilert and Swedish standard methods as was fecal streptococcus and enterococcus detection by both the Enterolert and Swedish standard methods.
Beauvericin Production by
Fusarium
Species
Authors: Logrieco A. , Moretti A. , Castella G. , Kostecki M. , Golinski P. , Ritieni A. , Chelkowski J.
Published: December 2019

Beauvericin is a cyclohexadepsipeptide mycotoxin which has insecticidal properties and which can induce apoptosis in mammalian cells. Beauvericin is produced by some entomo- and phytopathogenic
Fusarium
species (
Fusarium proliferatum
,
F. semitectum
, and
F. subglutinans
) and occurs naturally on corn and corn-based foods and feeds infected by
Fusarium
spp. We tested 94
Fusarium
isolates belonging to 25 taxa, 21 in 6 of the 12 sections of the
Fusarium
genus and 4 that have been described recently, for the ability to produce beauvericin. Beauvericin was produced by the following species (with the number of toxigenic strains compared with the number of tested strains given in parentheses):
Fusarium acuminatum
var.
acuminatum
(1 of 4),
Fusarium acuminatum
var.
armeniacum
(1 of 3),
F. anthophilum
(1 of 2),
F. avenaceum
(1 of 6),
F. beomiforme
(1 of 1),
F. dlamini
(2 of 2),
F. equiseti
(2 of 3),
F. longipes
(1 of 2),
F. nygamai
(2 of 2),
F. oxysporum
(4 of 7),
F. poae
(4 of 4),
F. sambucinum
(12 of 14), and
F. subglutinans
(3 of 3). These results indicate that beauvericin is produced by many species in the genus
Fusarium
and that it may be a contaminant of cereals other than maize.
Specific Ribosomal DNA Sequences from Diverse Environmental Settings Correlate with Experimental Contaminants
Authors: Tanner Michael A. , Goebel Brett M. , Dojka Michael A. , Pace Norman R.
Published: December 2019

Phylogenetic analysis of 16S ribosomal DNA (rDNA) clones obtained by PCR from uncultured bacteria inhabiting a wide range of environments has increased our knowledge of bacterial diversity. One possible problem in the assessment of bacterial diversity based on sequence information is that PCR is exquisitely sensitive to contaminating 16S rDNA. This raises the possibility that some putative environmental rRNA sequences in fact correspond to contaminant sequences. To document potential contaminants, we cloned and sequenced PCR-amplified 16S rDNA fragments obtained at low levels in the absence of added template DNA. 16S rDNA sequences closely related to the genera
Duganella
(formerly
Zoogloea
),
Acinetobacter
,
Stenotrophomonas
,
Escherichia
,
Leptothrix
, and
Herbaspirillum
were identified in contaminant libraries and in clone libraries from diverse, generally low-biomass habitats. The rRNA sequences detected possibly are common contaminants in reagents used to prepare genomic DNA. Consequently, their detection in processed environmental samples may not reflect environmentally relevant organisms.
Purification and Characterization of Laccase from
Chaetomium thermophilium
and Its Role in Humification
Authors: Chefetz Benny , Chen Yona , Hadar Yitzhak
Published: December 2019

Chaetomium thermophilium
was isolated from composting municipal solid waste during the thermophilic stage of the process.
C. thermophilium
, a cellulolytic fungus, exhibited laccase activity when it was grown at 45°C both in solid media and in liquid media. Laccase activity reached a peak after 24 h in liquid shake culture. Laccase was purified by ultrafiltration, anion-exchange chromatography, and affinity chromatography. The purified enzyme was identified as a glycoprotein with a molecular mass of 77 kDa and an isoelectric point of 5.1. The laccase was stable for 1 h at 70°C and had half-lives of 24 and 12 h at 40 and 50°C, respectively. The enzyme was stable at pH 5 to 10, and the optimum pH for enzyme activity was 6. The purified laccase efficiently catalyzed a wide range of phenolic substrates but not tyrosine. The highest levels of affinity were the levels of affinity to syringaldazine and hydroxyquinone. The UV-visible light spectrum of the purified laccase had a peak at 604 nm (i.e., Cu type I), and the activity was strongly inhibited by Cu-chelating agents. When the hydrophobic acid fraction (the humic fraction of the water-soluble organic matter obtained from municipal solid waste compost) was added to a reaction assay mixture containing laccase and guaiacol, polymerization took place and a soluble polymer was formed.
C. thermophilium
laccase, which is produced during the thermophilic stage of composting, can remain active for a long period of time at high temperatures and alkaline pH values, and we suggest that this enzyme is involved in the humification process during composting.
Biotransformation of the Major Fungal Metabolite 3,5-Dichloro-
p
-Anisyl Alcohol under Anaerobic Conditions and Its Role in Formation of Bis(3,5-Dichloro-4-Hydroxyphenyl)methane
Authors: Verhagen Frank J. M. , Swarts Henk J. , Wijnberg Joannes B. P. A. , Field Jim A.
Published: December 2019

Higher fungi have a widespread capacity for biosynthesis of organohalogens. Commonly occurring chloroaromatic fungal metabolites can end up in anaerobic microniches at the boundary of fungal colonies and wetland soils. The aim of this study was to investigate the environmental fate of a major fungal metabolite, 3,5-dichloro-
p
-anisyl alcohol, under anaerobic conditions. This compound was incubated with methanogenic sludge to study its biotransformation reactions. Initially, 3,5-dichloro-
p
-anisyl alcohol was readily demethylated in stoichiometric quantities to 3,5-dichloro-4-hydroxybenzyl alcohol. The demethylated product was converted further via two routes: a biotic route leading to the formation of 3,5-dichloro-4-hydroxybenzoate and 2,6-dichlorophenol, as well as an abiotic route leading to the formation of bis(3,5-dichloro-4-hydroxyphenyl)methane. In the first route, the benzyl alcohol moiety on the aromatic ring was oxidized, giving 3,5-dichloro-4-hydroxybenzoate as a transient or accumulating product, depending on the type of methanogenic sludge used. In sludge previously adapted to low-molecular-weight lignin from straw, a part of the 3,5-dichloro-4-hydroxybenzoate was decarboxylated, yielding detectable levels of 2,6-dichlorophenol. In the second route, 3,5-dichloro-4-hydroxybenzyl alcohol dimerized, leading to the formation of a tetrachlorinated bisphenolic compound, which was identified as bis(3,5-dichloro-4-hydroxyphenyl)methane. Since formation of this dimer was also observed in incubations with autoclaved sludge spiked with 3,5-dichloro-4-hydroxybenzyl alcohol, it was concluded that its formation was due to an abiotic process. However, demethylation of the fungal metabolite by biological processes was a prerequisite for dimerization. The most probable reaction mechanism leading to the formation of the tetrachlorinated dimer in the absence of oxygen is presented, and the possible environmental implications of its natural occurrence are discussed.
Effect of Toxic Metals on Indigenous Soil β-Subgroup Proteobacterium Ammonia Oxidizer Community Structure and Protection against Toxicity by Inoculated Metal-Resistant Bacteria
Authors: Stephen John R. , Chang Yun-Juan , Macnaughton Sarah J. , Kowalchuk George A. , Leung Kam T. , Flemming Cissy A. , White David C.
Published: December 2019

Contamination of soils with toxic metals is a major problem on military, industrial, and mining sites worldwide. Of particular interest to the field of bioremediation is the selection of biological markers for the end point of remediation. In this microcosm study, we focus on the effect of addition of a mixture of toxic metals (cadmium, cobalt, cesium, and strontium as chlorides) to soil on the population structure and size of the ammonia oxidizers that are members of the beta subgroup of the
Proteobacteria
(β-subgroup ammonia oxidizers). In a parallel experiment, the soils were also treated by the addition of five strains of metal-resistant heterotrophic bacteria. Effects on nitrogen cycling were measured by monitoring the NH
3
and NH
4
+
levels in soil samples. The gene encoding the α-subunit of ammonia monooxygenase (
amo
A) was selected as a functional molecular marker for the β-subgroup ammonia oxidizing bacteria. Community structure comparisons were performed with clone libraries of PCR-amplified fragments of
amo
A recovered from contaminated and control microcosms for 8 weeks. Analysis was performed by restriction digestion and sequence comparison. The abundance of ammonia oxidizers in these microcosms was also monitored by competitive PCR. All
amo
A gene fragments recovered grouped with sequences derived from cultured
Nitrosospira
. These comprised four novel sequence clusters and a single unique clone. Specific changes in the community structure of β-subgroup ammonia oxidizers were associated with the addition of metals. These changes were not seen in the presence of the inoculated metal-resistant bacteria. Neither treatment significantly altered the total number of β-subgroup ammonia-oxidizing cells per gram of soil compared to untreated controls. Following an initial decrease in concentration, ammonia began to accumulate in metal-treated soils toward the end of the experiment.
Heterologous Expression of
Pleurotus eryngii
Peroxidase Confirms Its Ability To Oxidize Mn
2+
and Different Aromatic Substrates
Authors: Ruiz-Dueñas Francisco Javier , Martínez María Jesús , Martínez Ángel T.
Published: December 2019

A versatile ligninolytic peroxidase has been cloned from
Pleurotus eryngii
and its allelic variant MnPL2 expressed in
Aspergillus nidulans
, with properties similar to those of the mature enzyme from
P. eryngii
. These include the ability to oxidize Mn
2+
and aromatic substrates, confirming that this is a new peroxidase type sharing catalytic properties of lignin peroxidase and manganese peroxidase.
PCR Detection, Characterization, and Distribution of Virulence Genes in
Aeromonas
spp
Authors: Kingombe Cesar Isigidi Bin , Huys Geert , Tonolla Mauro , Albert M. John , Swings Jean , Peduzzi Raffaele , Jemmi Thomas
Published: December 2019

We found 73.1 to 96.9% similarity by aligning the cytolytic enterotoxin gene of
Aeromonas hydrophila
SSU (AHCYTOEN; GenBank accession no.
M84709
) against aerolysin genes of
Aeromonas
spp., suggesting the possibility of selecting common primers. Identities of 90 to 100% were found among the eight selected primers from those genes. Amplicons obtained from
Aeromonas
sp. reference strains by using specific primers for each gene or a cocktail of primers were 232 bp long. Of hybridization group 4/5A/5B (HG4/5A/5B), HG9, and HG12 or non-
Aeromonas
reference strains, none were positive. PCR-restriction fragment length polymorphism (PCR-RFLP) with
Hpa
II yielded three types of patterns. PCR-RFLP 1 contained two fragments (66 and 166 bp) found in HG6, HG7, HG8, HG10, and HG11. PCR-RFLP 2 contained three fragments (18, 66, and 148 bp) found in HG1, HG2, HG3, and HG11. PCR-RFLP 3, with four fragments (7, 20, 66, and 139 bp), was observed only in HG13. PCR-amplicon sequence analysis (PCR-ASA) revealed three main types. PCR-ASA 1 had 76 to 78% homology with AHCYTOEN and included strains in HG6, HG7, HG8, HG10, and HG11. PCR-ASA 2, with 82% homology, was found only in HG13. PCR-ASA 3, with 91 to 99% homology, contained the strains in HG1, HG2, HG3, and HG11. This method indicated that 37 (61%) of the 61 reference strains were positive with the primer cocktail master mixture, and 34 (58%) of 59 environmental isolates, 93 (66%) of 141 food isolates, and 100 (67%) of 150 clinical isolates from around the world carried a virulence factor when primers AHCF1 and AHCR1 were used. In conclusion, this PCR-based method is rapid, sensitive, and specific for the detection of virulence factors of
Aeromonas
spp. It overcomes the handicap of time-consuming biochemical and other DNA-based methods.
Quantification of Bias Related to the Extraction of DNA Directly from Soils
Authors: Frostegård Åsa , Courtois Sophie , Ramisse Vincent , Clerc Sylvie , Bernillon Dominique , Le Gall Francoise , Jeannin Pascale , Nesme Xavier , Simonet Pascal
Published: December 2019

In recent years, several protocols based on the extraction of nucleic acids directly from the soil matrix after lysis treatment have been developed for the detection of microorganisms in soil. Extraction efficiency has often been evaluated based on the recovery of a specific gene sequence from an organism inoculated into the soil. The aim of the present investigation was to improve the extraction, purification, and quantification of DNA derived from as large a portion of the soil microbial community as possible, with special emphasis placed on obtaining DNA from gram-positive bacteria, which form structures that are difficult to disrupt. Furthermore, we wanted to identify and minimize the biases related to each step in the procedure. Six soils, covering a range of pHs, clay contents, and organic matter contents, were studied. Lysis was carried out by soil grinding, sonication, thermal shocks, and chemical treatments. DNA was extracted from the indigenous microflora as well as from inoculated bacterial cells, spores, and hyphae, and the quality and quantity of the DNA were determined by gel electrophoresis and dot blot hybridization. Lysis efficiency was also estimated by microscopy and viable cell counts. Grinding increased the extracellular DNA yield compared with the yield obtained without any lysis treatment, but none of the subsequent treatments clearly increased the DNA yield. Phage λ DNA was inoculated into the soils to mimic the fate of extracellular DNA. No more than 6% of this DNA could be recovered from the different soils. The clay content strongly influenced the recovery of DNA. The adsorption of DNA to clay particles decreased when the soil was pretreated with RNA in order to saturate the adsorption sites. We also investigated different purification techniques and optimized the PCR methods in order to develop a protocol based on hybridization of the PCR products and quantification by phosphorimaging.
Quantifying Microbial Diversity: Morphotypes, 16S rRNA Genes, and Carotenoids of Oxygenic Phototrophs in Microbial Mats
Authors: Nübel Ulrich , Garcia-Pichel Ferran , Kühl Michael , Muyzer Gerard
Published: December 2019

We quantified the diversity of oxygenic phototrophic microorganisms present in eight hypersaline microbial mats on the basis of three cultivation-independent approaches. Morphological diversity was studied by microscopy. The diversity of carotenoids was examined by extraction from mat samples and high-pressure liquid chromatography analysis. The diversity of 16S rRNA genes from oxygenic phototrophic microorganisms was investigated by extraction of total DNA from mat samples, amplification of 16S rRNA gene segments from cyanobacteria and plastids of eukaryotic algae by phylum-specific PCR, and sequence-dependent separation of amplification products by denaturing-gradient gel electrophoresis. A numerical approach was introduced to correct for crowding the results of chromatographic and electrophoretic analyses. Diversity estimates typically varied up to twofold among mats. The congruence of richness estimates and Shannon-Weaver indices based on numbers and proportional abundances of unique morphotypes, 16S rRNA genes, and carotenoids unveiled the underlying diversity of oxygenic phototrophic microorganisms in the eight mat communities studied.
Lla
FI, a Type III Restriction and Modification System in
Lactococcus lactis
Authors: Su Ping , Im Heejeong , Hsieh Hsiaoling , Kang’A Simon , Dunn Noel W.
Published: December 2019

We describe a type III restriction and modification (R/M) system,
Lla
FI, in
Lactococcus lactis. Lla
FI is encoded by a 12-kb native plasmid, pND801, harbored in
L. lactis
LL42-1. Sequencing revealed two adjacent open reading frames (ORFs). One ORF encodes a 680-amino-acid polypeptide, and this ORF is followed by a second ORF which encodes an 873-amino-acid polypeptide. The two ORFs appear to be organized in an operon. A homology search revealed that the two ORFs exhibited significant similarity to type III restriction (Res) and modification (Mod) subunits. The complete amino acid sequence of the Mod subunit of
Lla
FI was aligned with the amino acid sequences of four previously described type III methyltransferases. Both the N-terminal regions and the C-terminal regions of the Mod proteins are conserved, while the central regions are more variable. An S-adenosyl methionine (Ado-Met) binding motif (present in all adenine methyltransferases) was found in the N-terminal region of the Mod protein. The seven conserved helicase motifs found in the previously described type III R/M systems were found at the same relative positions in the
Lla
FI Res sequence.
Lla
FI has cofactor requirements for activity that are characteristic of the previously described type III enzymes. ATP and Mg
2+
are required for endonucleolytic activity; however, the activity is not strictly dependent on the presence of Ado-Met but is stimulated by it. To our knowledge, this is the first type III R/M system that has been characterized not just in lactic acid bacteria but also in gram-positive bacteria.
Chemical and Sensory Changes Associated with Microbial Flora of Mediterranean Boque (
Boops boops
) Stored Aerobically at 0, 3, 7, and 10°C
Authors: Koutsoumanis Konstantinos , Nychas George-John E.
Published: December 2019

The development of a microbial population and changes in the physicochemical and sensorial characteristics of Mediterranean boque (
Boops boops
), called gopa in Greece, stored aerobically at 0, 3, 7, and 10°C were studied. Pseudomonads and
Shewanella putrefaciens
were the dominant bacteria at the end of the storage period, regardless of the temperature tested. Enterobacteria and
Brochothrix thermosphacta
also grew, but their population density was always 2 to 3 log
10
CFU g
−1
less than that of pseudomonads. The concentration of potential indicators of spoilage, glucose and lactic acid, decreased while that of the α-amino groups increased during storage. The concentrations of these carbon sources also decreased on sterile fish blocks inoculated with strains isolated from fish microbial flora. The organic acid profile of sterile fish blocks inoculated with the above-mentioned bacteria and that of naturally spoiled fish differed significantly. An excellent correlation (
r
= −0.96) between log
10
counts of
S. putrefaciens
or
Pseudomonas
bacteria with freshness was observed in this study.
Exopolysaccharide-Producing Bacteria from Sugar Beets
Authors: Tallgren Antti H. , Airaksinen Ulla , von Weissenberg Robert , Ojamo Heikki , Kuusisto Juhani , Leisola Matti
Published: December 2019

Six hundred microorganisms were isolated from sugar beets collected from different parts of Finland to study their slime production. A total of 170 of them produced exopolysaccharides, of which 35% were heteropolysaccharides. The yield of heteropolysaccharides from sucrose was lower than that of dextrans. Five isolates, which were chosen for closer study, were identified as
Leuconostoc mesenteroides
(two species),
Rahnella aquatilis
(two species), and
Enterobacter amnigenus.
Combination of Fluorescent In Situ Hybridization and Microautoradiography—a New Tool for Structure-Function Analyses in Microbial Ecology
Authors: Lee Natuscka , Nielsen Per Halkjær , Andreasen Kjær Holm , Juretschko Stefan , Nielsen Jeppe Lund , Schleifer Karl-Heinz , Wagner Michael
Published: December 2019

A new microscopic method for simultaneously determining in situ the identities, activities, and specific substrate uptake profiles of individual bacterial cells within complex microbial communities was developed by combining fluorescent in situ hybridization (FISH) performed with rRNA-targeted oligonucleotide probes and microautoradiography. This method was evaluated by using defined artificial mixtures of
Escherichia coli
and
Herpetosiphon aurantiacus
under aerobic incubation conditions with added [
3
H]glucose. Subsequently, we were able to demonstrate the potential of this method by visualizing the uptake of organic and inorganic radiolabeled substrates ([
14
C]acetate, [
14
C]butyrate, [
14
C]bicarbonate, and
33
P
i
) in probe-defined populations from complex activated sludge microbial communities by using aerobic incubation conditions and anaerobic incubation conditions (with and without nitrate). For both defined cell mixtures and activated sludge, the method proved to be useful for simultaneous identification and analysis of the uptake of labeled substrates under the different experimental conditions used. Optimal results were obtained when fluorescently labeled oligonucleotides were applied prior to the microautoradiographic developing procedure. For single-cell resolution of FISH and microautoradiographic signals within activated sludge flocs, cryosectioned sample material was examined with a confocal laser scanning microscope. The combination of in situ rRNA hybridization techniques, cryosectioning, microautoradiography, and confocal laser scanning microscopy provides a unique opportunity for obtaining cultivation-independent insights into the structure and function of bacterial communities.
A Double-Selective Tissue Culture System for Isolation of Wild-Type Poliovirus from Sewage Applied in a Long-Term Environmental Surveillance
Authors: Manor Y. , Handsher R. , Halmut T. , Neuman M. , Abramovitz B. , Mates A. , Mendelson E.
Published: December 2019

We describe a simple, cost-efficient, double-selective method for isolation of wild-type poliovirus from sewage samples containing vaccine polioviruses and other enteroviruses, with a detection limit of 18 to 50 PFU per 1 to 2 liters of sewage. By this method we were able to process 1,700 sewage samples collected between 1991 and 1996, from which 10,472 plaques were isolated, 41 of them being identified as wild-type polioviruses.
Clinical and Veterinary Isolates of
Salmonella enterica
Serovar Enteritidis Defective in Lipopolysaccharide O-Chain Polymerization
Authors: Guard-Petter J. , Parker C. T. , Asokan K. , Carlson R. W.
Published: December 2019

Twelve human and chicken isolates of
Salmonella enterica
serovar Enteritidis belonging to phage types 4, 8, 13a, and 23 were characterized for variability in lipopolysaccharide (LPS) composition. Isolates were differentiated into two groups, i.e., those that lacked immunoreactive O-chain, termed rough isolates, and those that had immunoreactive O-chain, termed smooth isolates. Isolates within these groups could be further differentiated by LPS compositional differences as detected by gel electrophoresis and gas liquid chromatography of samples extracted with water, which yielded significantly more LPS in comparison to phenol-chloroform extraction. The rough isolates were of two types, the O-antigen synthesis mutants and the O-antigen polymerization (
wzy
) mutants. Smooth isolates were also of two types, one producing low-molecular-weight (LMW) LPS and the other producing high-molecular-weight (HMW) LPS. To determine the genetic basis for the O-chain variability of the smooth isolates, we analyzed the effects of a null mutation in the O-chain length determinant gene,
wzz
(
cld
) of serovar Typhimurium. This mutation results in a loss of HMW LPS; however, the LMW LPS of this mutant was longer and more glucosylated than that from clinical isolates of serovar Enteritidis. Cluster analysis of these data and of those from two previously characterized isogenic strains of serovar Enteritidis that had different virulence attributes indicated that glucosylation of HMW LPS (via
oafR
function) is variable and results in two types of HMW structures, one that is highly glucosylated and one that is minimally glucosylated. These results strongly indicate that naturally occurring variability in
wzy
,
wzz
, and
oafR
function can be used to subtype isolates of serovar Enteritidis during epidemiological investigations.
An Alkane-Responsive Expression System for the Production of Fine Chemicals
Authors: Panke Sven , Meyer Andreas , Huber Caroline M. , Witholt Bernard , Wubbolts Marcel G.
Published: December 2019

Membrane-located monooxygenase systems, such as the
Pseudomonas putida
mt-2-derived xylene oxygenase, are attractive for challenging transformations of apolar compounds, including enantiospecific epoxidations, but are difficult to synthesize at levels that are useful for application to biotechnological processes. In order to construct efficient biocatalysis strains, we utilized the alkane-responsive regulatory system of the OCT plasmid-located
alk
genes of
Pseudomonas oleovorans
GPo1, a very attractive system for recombinant biotransformation processes. Determination of the nucleotide sequence of
alkS
, whose activated gene product positively regulates the transcription of the structural genes
alkBFGHJKL
, on a 3.7-kb
Sal
I-
Hpa
I OCT plasmid fragment was completed, and the N-terminal amino acid sequence of an AlkS-LacZ fusion protein was found to be consistent with the predicted DNA sequence. The
alkS
gene and the
alkBp
promoter were assembled into a convenient alkane-responsive genetic expression cassette which allowed expression of the xylene oxygenase genes in a recombinant
Escherichia coli
strain at a specific activity of 91 U per g (dry weight) of cells when styrene was the substrate. This biocatalyst was used to produce (
S
)-styrene oxide in two-liquid-phase cultures. Volumetric productivities of more than 2 g of styrene oxide per h per liter of aqueous phase were obtained; these values represented a fivefold improvement compared with previous results.