bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation and Characterization of Phenol-Degrading Denitrifying Bacteria
Authors: van Schie Paula M. , Young L. Y. 
Published: December 2019

ABSTRACT

Phenol is a man-made as well as a naturally occurring aromatic compound and an important intermediate in the biodegradation of natural and industrial aromatic compounds. Whereas many microorganisms that are capable of aerobic phenol degradation have been isolated, only a few phenol-degrading anaerobic organisms have been described to date. In this study, three novel nitrate-reducing microorganisms that are capable of using phenol as a sole source of carbon were isolated and characterized. Phenol-degrading denitrifying pure cultures were obtained by enrichment culture from anaerobic sediments obtained from three different geographic locations, the East River in New York, N.Y., a Florida orange grove, and a rain forest in Costa Rica. The three strains were shown to be different from each other based on physiologic and metabolic properties. Even though analysis of membrane fatty acids did not result in identification of the organisms, the fatty acid profiles were found to be similar to those of
Azoarcus
species. Sequence analysis of 16S ribosomal DNA also indicated that the phenol-degrading isolates were closely related to members of the genus
Azoarcus
. The results of this study add three new members to the genus
Azoarcus
, which previously comprised only nitrogen-fixing species associated with plant roots and denitrifying toluene degraders.
Detection of Hemolysin Variants of Shiga Toxin-Producing
Escherichia coli
by PCR and Culture on VancomycinCefixime-Cefsulodin Blood Agar
Authors: Lehmacher Anselm , Meier Heidi , Aleksic Stojanka , Bockemühl Jochen 
Published: December 2019

ABSTRACT

The presence of a hemolysin-encoding gene,
elyA
or
hlyA
, from Shiga toxin-producing
Escherichia coli
(STEC) was detected by PCR in each of 95 strains tested. PCR products of
elyA
from human STEC isolates of serovars frequently detected in Germany, such as O157:H−, O103:H2, O103:H−, O26:H11, and O26:H−, showed nucleotide sequences identical to previously reported ones for O157:H7 and O111:H− strains. Compared to them, four
elyA
amplicons derived from human isolates of rare STEC serovars showed identity of about 98% but lacked an
Alu
I restriction site. However, the nucleotide sequence of an amplicon derived from a porcine O138:K81:H− STEC strain was identical to the corresponding region of
hlyA
, encoding alpha-hemolysin, from
E. coli
. This
hlyA
amplicon showed 68% identity with the nucleotide sequence of the corresponding
elyA
fragment. It differed from the
elyA
PCR product in restriction fragments generated by
Alu
I,
Eco
RI, and
Mlu
I. Of the 95 representative STEC strains, 88 produced hemolysin on blood agar supplemented with vancomycin (30 mg/liter), cefixime (20 μg/liter), and cefsulodin (3 mg/liter) (BVCC). The lowest added numbers of two to six STEC CFU per g of stool or per ml of raw milk were detectable on BVCC plates after seeding of the preenrichment broth, modified tryptic soy broth (mTSB) supplemented with novobiocin (10 mg/liter), with 16 STEC strains. These strains represented the seven prevailing serovars diagnosed from German patients. However, with ground-beef samples, PCR was essential to identify the lowest added numbers of two to six STEC CFU among colonies of hemolyzing
Enterobacteriaceae
, such as
Serratia
spp. and alpha-hemolysin-producing
E. coli
. We conclude that preenrichment of stool and food samples in mTSB for 6 h followed by overnight culturing on BVCC is a simple method for the isolation and presumptive identification of STEC.
Construction and Use of an
ipb
DNA Module To Generate
Pseudomonas
Strains with Constitutive Trichloroethene and Isopropylbenzene Oxidation Activity
Authors: Berendes Frank , Sabarth Nicolas , Averhoff Beate , Gottschalk Gerhard 
Published: December 2019

ABSTRACT

Pseudomonas
sp. strain JR1 exhibits trichloroethene (TCE) oxidation activity with isopropylbenzene (IPB) as the inducer substrate. We previously reported the genes encoding the first three enzymes of the IPB-degradative pathway (
ipbA1
,
ipbA2
,
ipbA3
,
ipbA4
,
ipbB
, and
ipbC
) and identified the initial IPB dioxygenase (IpbA1A2A3A4) as responsible for TCE cooxidation (U. Pflugmacher, B. Averhoff, and G. Gottschalk, Appl. Environ. Microbiol. 62:3967–3977, 1996). Primer extension analyses revealed multiple transcriptional start points located upstream of the translational initiation codon of
ipbA1
. The transcription from these start sites was found to be IPB dependent. Thirty-one base pairs upstream of the first transcriptional start point tandemly repeated DNA sequences overlapping the −35 region of a putative ς
70
promoter were found. These repeats exhibit significant sequence similarity to the operator-promoter region of the
xyl meta
operon in
Pseudomonas putida
, which is required for the binding of XylS, a regulatory protein of the XylS (also called AraC) family. These similarities suggest that the transcription of the IPB dioxygenase genes is modulated by a regulatory protein of the XylS/AraC family. The construction of an
ipb
DNA module devoid of this
ipb
operator-promoter region and the stable insertion of this DNA module into the genomes of different
Pseudomonas
strains resulted in pseudomonads with constitutive IPB and TCE oxidation activities. Constitutive TCE oxidation of two such
Pseudomonas
hybrid strains, JR1A::
ipb
and CBS-3::
ipb
, was found to be stable for more than 120 generations in antibiotic-free medium. Evaluation of constitutive TCE degradation rates revealed that continuous cultivation of strain JR1A::
ipb
resulted in a significant increase in rates of TCE degradation.
Small-Scale DNA Sample Preparation Method for Field PCR Detection of Microbial Cells and Spores in Soil
Authors: Kuske Cheryl R. , Banton Kaysie L. , Adorada Dante L. , Stark Peter C. , Hill Karen K. , Jackson Paul J. 
Published: December 2019

ABSTRACT

Efficient, nonselective methods to obtain DNA from the environment are needed for rapid and thorough analysis of introduced microorganisms in environmental samples and for analysis of microbial community diversity in soil. A small-scale procedure to rapidly extract and purify DNA from soils was developed for in-the-field use. Amounts of DNA released from bacterial vegetative cells, bacterial endospores, and fungal conidia were compared by using hot-detergent treatment, freeze-thaw cycles, and bead mill homogenization. Combining a hot-detergent treatment with bead mill homogenization gave the highest DNA yields from all three microbial cell types and provided DNA from the broadest range of microbial groups in a natural soil community. Only the bead mill homogenization step was effective for DNA extraction from
Bacillus globigii
(
B. subtilis
subsp.
niger
) endospores or
Fusarium moniliforme
conidia. The hot-detergent–bead mill procedure was simplified and miniaturized. By using this procedure and small-scale, field-adapted purification and quantification procedures, DNA was prepared from four different soils seeded with
Pseudomonas putida
cells or
B. globigii
spores. In a New Mexico soil, seeded bacterial targets were detected with the same sensitivity as when assaying pure bacterial DNA (2 to 20 target gene copies in a PCR mixture). The detection limit of
P. putida
cells and
B. globigii
spores in different soils was affected by the amount of background DNA in the soil samples, the physical condition of the DNA, and the amount of DNA template used in the PCR.
Molecular Analysis of Bacterial Communities in a Three-Compartment Granular Activated Sludge System Indicates Community-Level Control by Incompatible Nitrification Processes
Authors: Holben William E. , Noto Kazuhiko , Sumino Tatsuo , Suwa Yuichi 
Published: December 2019

ABSTRACT

Bacterial community structure and the predominant nitrifying activities and populations in each compartment of a three-compartment activated sludge system were determined. Each compartment was originally inoculated with the same activated sludge community entrapped in polyethylene glycol gel granules, and ammonium nitrogen was supplied to the system in an inorganic salts solution at a rate of 5.0 g of N liter of granular activated sludge
−1
day
−1
. After 150 days of operation, the system was found to comprise a series of sequential nitrifying reactions (K. Noto, T. Ogasawara, Y. Suwa, and T. Sumino, Water Res. 32:769–773, 1998), presumably mediated by different bacterial populations. Activity data showed that all NH
4
-N was completely oxidized in compartments one and two (approximately half in each), but no significant nitrite oxidation was observed in these compartments. In contrast, all available nitrite was oxidized to nitrate in compartment three. To study the microbial populations and communities in this system, total bacterial DNA isolated from each compartment was analyzed for community structure based on the G+C contents of the component populations. Compartment one showed dominant populations having 50 and 67% G+C contents. Compartment two was similar in structure to compartment one. The bacterial community in compartment three had dominant populations with 62 and 67% G+C contents and retained the 50% G+C content population only at a greatly diminished level. The 50% G+C content population from compartment one hybridized strongly with
amo
(ammonia monooxygenase) and
hao
(hydroxylamine oxidoreductase) gene probes from
Nitrosomonas europaea
. However, the 50% G+C content population from compartment two hybridized strongly with the
hao
probe but only weakly with the
amo
probe, suggesting that the predominant ammonia-oxidizing populations in compartments one and two might be different. Since different activities and populations come to dominate in each compartment from an identical inoculum, it appears that the nitrification processes may be somewhat incompatible, resulting in a series of sequential reactions and different communities in this three-compartment system.
Spatial and Temporal Variation of Phenanthrene-Degrading Bacteria in Intertidal Sediments
Authors: Berardesco Gina , Dyhrman Sonya , Gallagher Eugene , Shiaris Michael P. 
Published: December 2019

ABSTRACT

Phenanthrene-degrading bacteria were isolated from a 1-m
2
intertidal sediment site in Boston Harbor. Samples were taken six times over 2 years. A total of 432 bacteria were isolated and characterized by biochemical testing. When clustered on the basis of phenotypic characteristics, the isolates could be separated into 68 groups at a similarity level of approximately 70%. Several groups (a total of 200 isolates) corresponded to well-characterized species belonging the genera
Vibrio
and
Pseudomonas
. Only 51 of the 437 isolates (<11.7% of the total) hybridized to a DNA probe that encodes the upper pathway of naphthalene and phenanthrene degradation in
Pseudomonas putida
NCIB 9816. A cluster analysis indicated that the species composition of the phenanthrene-degrading community changed significantly from sampling date to sampling date. At one sampling time, 12 6-mm-diameter core subsamples were taken within the 1-m
2
site to determine the spatial variability of the degrading communities. An analysis of molecular variance, performed with the phenotypic characteristics, indicated that only 6% of the variation occurred among the 12 subsamples, suggesting that the subsamples were almost identical in composition. We concluded that the communities of phenanthrene-degrading bacteria in the sediments are very diverse, that the community structure undergoes significant change with time but does not vary significantly on a spatial scale of centimeters, and that the predominant genes that encode phenanthrene degradation in the communities are not well-characterized.
Aerobic Mineralization of 2,6-Dichlorophenol by
Ralstonia
sp. Strain RK1
Authors: Steinle Patrick , Stucki Gerhard , Stettler Rolf , Hanselmann Kurt W. 
Published: December 2019

ABSTRACT

A new aerobic bacterium was isolated from the sediment of a freshwater pond close to a contaminated site at Amponville (France). It was enriched in a fixed-bed reactor fed with 2,6-dichlorophenol (2,6-DCP) as the sole carbon and energy source at pH 7.5 and room temperature. The degradation of 2,6-DCP followed Monod kinetics at low initial concentrations. At concentrations above 300 μM (50 mg · liter
−1
), 2,6-DCP increasingly inhibited its own degradation. The base sequence of the 16S ribosomal DNA allowed us to assign the bacterium to the genus
Ralstonia
(formerly
Alcaligenes
). The substrate spectrum of the bacterium includes toluene, benzene, chlorobenzene, phenol, and all four
ortho
- and
para
-substituted mono- and dichlorophenol isomers. Substituents other than chlorine prevented degradation. The capacity to degrade 2,6-DCP was examined in two fixed-bed reactors. The microbial population grew on and completely mineralized 2,6-DCP at 2,6-DCP concentrations up to 740 μM in continuous reactor culture supplied with H
2
O
2
as an oxygen source. Lack of peroxide completely stopped further degradation of 2,6-DCP. Lowering the acid-neutralizing capacity of the medium to 1/10th the original capacity led to a decrease in the pH of the effluent from 7 to 6 and to a significant reduction in the degradation activity. A second fixed-bed reactor successfully removed low chlorophenol concentrations (20 to 26 μM) with hydraulic residence times of 8 to 30 min.
Capillary Electrophoresis Measurements of Electrophoretic Mobility for Colloidal Particles of Biological Interest
Authors: Glynn J. R. , Belongia B. M. , Arnold R. G. , Ogden K. L. , Baygents J. C. 
Published: December 2019

ABSTRACT

The electrophoretic mobilities of three bacterial strains were investigated by capillary electrophoresis (CE) and were compared with results obtained by microelectrophoresis (ME). The CE measurements yielded bimodal electropherograms for two of the strains, thus illustrating for the first time that surface charge variations within a monoclonal population can be probed by CE. Intrapopulation variations were not detected by ME. The mobilities of three chemically distinct types of latex microspheres were also measured. Differences between the mean mobilities obtained by CE and ME were not statistically significant (
P
≤ 0.50); the standard deviations of the CE measurements were typically 2 to 10 times smaller than those obtained by comparable ME measurements. The reproducibility of CE permitted batch-to-batch mobility variations to be probed for the bacteria (one of the strains exhibited such variations), and aggregation was evident in one of the latex suspensions. These effects were not measurable with ME.
Effectiveness of SYTOX Green Stain for Bacterial Viability Assessment
Authors: Lebaron P. , Catala P. , Parthuisot N. 
Published: December 2019

ABSTRACT

The effectiveness of SYTOX Green nucleic acid stain for measuring bacterial viability was tested on starved populations of
Escherichia coli
and
Salmonella typhimurium
. This stain underestimates the fraction of dead cells within starved populations containing cells with damaged nucleic acids or membranes. Its application to natural samples should be considered with caution.
Insecticidal Activity of
Bacillus laterosporus
Authors: Orlova Margarita V. , Smirnova Tatyana A. , Ganushkina Lyudmila A. , Yacubovich Victoria Y. , Azizbekyan Roudolf R. 
Published: December 2019

ABSTRACT

The
Bacillus laterosporus
strains 921 and 615 were shown to have toxicity for larvae of the mosquitoes
Aedes aegypti
,
Anopheles stephensi
, and
Culex pipiens
. The larvicidal activity of
B. laterosporus
was associated with spores and crystalline inclusions. Purified
B. laterosporus
615 crystals were highly toxic for
Aedes aegypti
and
Anopheles stephensi
.
Microbial Community Changes in a Perturbed Agricultural Soil Investigated by Molecular and Physiological Approaches
Authors: Øvreås Lise , Jensen Sigmund , Daae Frida Lise , Torsvik Vigdis 
Published: December 2019

ABSTRACT
Changes in soil microbial activity and diversity after incubation either with nitrogen or with a mixture of methane and air were examined. The perturbation by methane and air were characterized in detail and led to reduced diversity and enrichment of methanotrophs which were identified by denaturing gradient gel electrophoresis and 16S rRNA sequencing.
Genetic Diversity of
nifH
Gene Sequences in
Paenibacillus azotofixans
Strains and Soil Samples Analyzed by Denaturing Gradient Gel Electrophoresis of PCR-Amplified Gene Fragments
Authors: Rosado Alexandre S. , Duarte Gabriela F. , Seldin Lucy , Van Elsas Jan Dirk 
Published: December 2019

ABSTRACT

The diversity of dinitrogenase reductase gene (
nifH
) fragments in
Paenibacillus azotofixans
strains was investigated by using molecular methods. The partial
nifH
gene sequences of eight
P. azotofixans
strains, as well as one strain each of the close relatives
Paenibacillus durum
,
Paenibacillus polymyxa
, and
Paenibacillus macerans
, were amplified by PCR by using degenerate primers and were characterized by DNA sequencing. We found that there are two
nifH
sequence clusters, designated clusters I and II, in
P. azotofixans
. The data further indicated that there was sequence divergence among the
nifH
genes of
P. azotofixans
strains at the DNA level. However, the gene products were more conserved at the protein level. Phylogenetic analysis showed that all
nifH
cluster II sequences were similar to the alternative (
anf
) nitrogenase sequence. A nested PCR assay for the detection of
nifH
(cluster I) of
P. azotofixans
was developed by using the degenerate primers as outer primers and two specific primers, designed on the basis of the sequence information obtained, as inner primers. The specificity of the inner primers was tested with several diazotrophic bacteria, and PCR revealed that these primers are specific for the
P. azotofixans nifH
gene. A GC clamp was attached to one inner primer, and a denaturing gradient gel electrophoresis (DGGE) protocol was developed to study the genetic diversity of this region of
nifH
in
P. azotofixans
strains, as well as in soil and rhizosphere samples. The results revealed sequence heterogeneity among different
nifH
genes. Moreover,
nifH
is probably a multicopy gene in
P. azotofixans
. Both similarities and differences were detected in the
P. azotofixans nifH
DGGE profiles generated with soil and rhizosphere DNAs. The DGGE assay developed here is reproducible and provides a rapid way to assess the intraspecific genetic diversity of an important functional gene in pure cultures, as well as in environmental samples.
Acetoacetyl Coenzyme A Reductase and Polyhydroxybutyrate Synthesis in
Rhizobium
(
Cicer
) sp. Strain CC 1192
Authors: Chohan Shahid N. , Copeland Les 
Published: December 2019

ABSTRACT

Biochemical controls that regulate the biosynthesis of poly-3-hydroxybutyrate (PHB) were investigated in
Rhizobium
(
Cicer
) sp. strain CC 1192. This species is of interest for studying PHB synthesis because the polymer accumulates to a large extent in free-living cells but not in bacteroids during nitrogen-fixing symbiosis with chickpea (
Cicer arietinum
L.) plants. Evidence is presented that indicates that CC 1192 cells retain the enzymic capacity to synthesize PHB when they differentiate from the free-living state to the bacteroid state. This evidence includes the incorporation by CC 1192 bacteroids of radiolabel from [
14
C]malate into 3-hydroxybutyrate which was derived by chemically degrading insoluble material from bacteroid pellets. Furthermore, the presence of an NADPH-dependent acetoacetyl coenzyme A (CoA) reductase, which was specific for
R
-(−)-3-hydroxybutyryl-CoA and NADP
+
in the oxidative direction, was demonstrated in extracts from free-living and bacteroid cells of CC 1192. Activity of this enzyme in the reductive direction appeared to be regulated at the biochemical level mainly by the availability of substrates. The CC 1192 cells also contained an NADH-specific acetoacetyl-CoA reductase which oxidized
S
-(+)-3-hydroxybutyryl-CoA. A membrane preparation from CC 1192 bacteroids readily oxidized NADH but not NADPH, which is suggested to be a major source of reductant for nitrogenase. Thus, a high ratio of NADPH to NADP
+
, which could enhance delivery of reductant to nitrogenase, could also favor the reduction of acetoacetyl-CoA for PHB synthesis. This would mean that fine controls that regulate the partitioning of acetyl-CoA between citrate synthase and 3-ketothiolase are important in determining whether PHB accumulates.
Influence of a Cell-Wall-Associated Protease on Production of α-Amylase by
Bacillus subtilis
Authors: Stephenson Keith , Harwood Colin R. 
Published: December 2019

ABSTRACT

AmyL, an extracellular α-amylase from
Bacillus licheniformis
, is resistant to extracellular proteases secreted by
Bacillus subtilis
during growth. Nevertheless, when AmyL is produced and secreted by
B. subtilis
, it is subject to considerable cell-associated proteolysis. Cell-wall-bound proteins CWBP52 and CWBP23 are the processed products of the
B. subtilis wprA
gene. Although no activity has been ascribed to CWBP23, CWBP52 exhibits serine protease activity. Using a strain encoding an inducible
wprA
gene, we show that a product of
wprA
, most likely CWBP52, is involved in the posttranslocational stability of AmyL. A construct in which
wprA
is not expressed exhibits an increased yield of α-amylase. The potential role of
wprA
in protein secretion is discussed, together with implications for the use of
B. subtilis
and related bacteria as hosts for the secretion of heterologous proteins.
A Novel β-
N
-Acetylglucosaminidase from
Streptomyces thermoviolaceus
OPC-520: Gene Cloning, Expression, and Assignment to Family 3 of the Glycosyl Hydrolases
Authors: Tsujibo Hiroshi , Hatano Naoya , Mikami Tadahisa , Hirasawa Ayako , Miyamoto Katsushiro , Inamori Yoshihiko 
Published: December 2019

ABSTRACT

A β-
N
-acetylglucosaminidase gene (
nagA
) of
Streptomyces thermoviolaceus
OPC-520 was cloned in
Streptomyces lividans
66. The nucleotide sequence of the gene, which encodes NagA, revealed an open reading frame of 1,896 bp, encoding a protein with an
M
r
of 66,329. The deduced primary structure of NagA was confirmed by comparison with the N-terminal amino acid sequence of the cloned β-
N
-acetylglucosaminidase expressed by
S. lividans
. The enzyme shares no sequence similarity with the classical β-
N
-acetylglucosaminidases belonging to family 20. However, NagA, which showed no detectable β-glucosidase activity, revealed homology with microbial β-glucosidases belonging to family 3; in particular, striking homology with the active-site regions of β-glucosidases was observed. Thus, the above-mentioned results indicate that NagA from
S. thermoviolaceus
OPC-520 is classified as a family 3 glycosyl hydrolase. The enzyme activity was optimal at 60°C and pH 5.0, and the apparent

K
m

and
V
max
values for
p
-nitrophenyl-β-
N
-acetylglucosamine were 425.7 μM and 24.8 μmol min
−1
mg of protein
−1
, respectively.