bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT
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

ABSTRACT
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT
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

ABSTRACT

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

ABSTRACT

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.