bluuebunny/crossref_metadata_2025_split huggingface dataset:
Microcosm Enrichment of Biphenyl-Degrading Microbial Communities from Soils and Sediments
Authors: Wagner-Döbler I. , Bennasar A. , Vancanneyt M. , Strömpl C. , Brümmer I. , Eichner C. , Grammel I. , Moore E. R. B. 
Published: December 2019

ABSTRACT

A microcosm enrichment approach was employed to isolate bacteria which are representative of long-term biphenyl-adapted microbial communities. Growth of microorganisms was stimulated by incubating soil and sediment samples from polluted and nonpolluted sites with biphenyl crystals. After 6 months, stable population densities between 8 × 10
9
and 2 × 10
11
CFU/ml were established in the microcosms, and a large percentage of the organisms were able to grow on biphenyl-containing minimal medium plates. A total of 177 biphenyl-degrading strains were subsequently isolated and characterized by their ability to grow on biphenyl in liquid culture and to accumulate a yellow
meta
cleavage product when they were sprayed with dihydroxybiphenyl. Isolates were identified by using a polyphasic approach, including fatty acid methyl ester (FAME) analysis, 16S rRNA gene sequence comparison, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins, and genomic fingerprinting based on sequence variability in the 16S-23S ribosomal DNA intergenic spacer region. In all of the microcosms, isolates identified as
Rhodococcus opacus
dominated the cultivable microbial community, comprising a cluster of 137 isolates with very similar FAME profiles (Euclidean distances, <10) and identical 16S rRNA gene sequences. The
R. opacus
isolates from the different microcosms studied could not be distinguished from each other by any of the fingerprint methods used. In addition, three other FAME clusters were found in one or two of the microcosms analyzed; these clusters could be assigned to
Alcaligenes
sp.,
Terrabacter
sp., and
Bacillus thuringiensis
on the basis of their FAME profiles and/or comparisons of the 16S rRNA gene sequences of representatives. Thus, the microcosm enrichments were strongly dominated by gram-positive bacteria, especially the species
R. opacus
, independent of the pollution history of the original sample.
R. opacus
, therefore, is a promising candidate for development of effective long-term inocula for polychlorinated biphenyl bioremediation.
A Novel Insecticidal Toxin from
Photorhabdus luminescens
, Toxin Complex a (Tca), and Its Histopathological Effects on the Midgut of
Manduca sexta
Authors: Blackburn Michael , Golubeva Elena , Bowen David , Ffrench-Constant Richard H. 
Published: December 2019

ABSTRACT

Photorhabdus luminescens
is a bacterium which is mutualistic with entomophagous nematodes and which secretes high-molecular-weight toxin complexes following its release into the insect hemocoel upon nematode invasion. Thus, unlike other protein toxins from
Bacillus thuringiensis
(δ-endotoxins and Vip’s),
P. luminescens
toxin (Pht) normally acts from within the insect hemocoel. Unexpectedly, therefore, the toxin complex has both oral and injectable activities against a wide range of insects. We have recently fractionated the protein toxin and shown it to consist of several native complexes, the most abundant of which we have termed Toxin complex a (Tca). This complex is highly active against the lepidopteran
Manduca sexta
. In view of the difference in the normal mode of delivery of
P. luminescens
toxin and the apparent communality in the histopathological effects of other gut-active toxins from
B. thuringiensis
, as well as cholesterol oxidase, we were interested in investigating the effects of purified Tca protein on larvae of
M. sexta
. Here we report that the histopathology of the
M. sexta
midgut is similar to that for other novel midgut-active toxins. Following oral ingestion of Tca by
M. sexta
, we observed an acceleration in the blebbing of the midgut epithelium into the gut lumen and eventual lysis of the epithelium. The midgut shows a similar histopathology following injection of Tca into the insect hemocoel. These results not only show that Tca is a highly active oral insecticide but also confirm the similar histopathologies of a range of very different gut-active toxins, despite presumed differences in modes of action and/or delivery. The implications for the mode of action of Tca are discussed.
Heat Shock Response in the Thermophilic Enteric Yeast
Arxiozyma telluris
Authors: Deegenaars Michelle L. , Watson Kenneth 
Published: December 2019

ABSTRACT

Heat stress tolerance was examined in the thermophilic enteric yeast
Arxiozyma telluris
. Heat shock acquisition of thermotolerance and synthesis of heat shock proteins hsp 104, hsp 90, hsp 70, and hsp 60 were induced by a mild heat shock at temperatures from 35 to 40°C for 30 min. The results demonstrate that a yeast which occupies a specialized ecological niche exhibits a typical heat shock response.
Unstable Expression and Thermal Instability of a Species-Specific Cell Surface Epitope Associated with a 66-Kilodalton Antigen Recognized by Monoclonal Antibody EM-7G1 within Serotypes of
Listeria monocytogenes
Grown in Nonselective and Selective Broths
Authors: Nannapaneni Ramakrishna , Story Robert , Bhunia Arun K. , Johnson Michael G. 
Published: December 2019

ABSTRACT

Conditions that resulted in unstable expression and heat instability of a cell surface epitope associated with a 66-kDa antigen in
Listeria monocytogenes
serotypes were identified with the probe monoclonal antibody (MAb) EM-7G1 in an enzyme-linked immunosorbent assay. This epitope appeared to be absent in three serotypes (serotypes 3b, 4a, and 4c), which did not react with MAb EM-7G1 irrespective of the enrichment broth tested. The remaining 10 serotypes were detected by MAb EM-7G1 only when cells were grown in nonselective brain heart infusion broth (BHI) or selective
Listeria
enrichment broth (LEB). When cells were grown in
Listeria
repair broth (LRB), only 6 of the 13 serotypes were detected by MAb EM-7G1, and recognition of serogroup 4 was completely lost. None of the 13 serotypes was detected by MAb EM-7G1 when cells were grown in two other commonly used
Listeria
-selective media, UVM1 broth and Fraser broth (FRB), indicating that possible loss of epitope expression occurred under these conditions. MAb EM-7G1 maintained species specificity without cross-reacting with live or heat-killed cells of six other
Listeria
spp. (
Listeria ivanovii
,
Listeria innocua
,
Listeria seeligeri
,
Listeria welshimeri
,
Listeria grayi
, and
Listeria murrayi
) irrespective of the enrichment conditions tested. Due to heat instability of the cell surface epitope when it was exposed to 80 or 100°C for 20 min, MAb EM-7G1 is suitable for detection of live cells of
L. monocytogenes
in BHI or LEB but not in LRB, UVM1, or FRB enrichment medium.
Localization and Solubilization of the Iron(III) Reductase of
Geobacter sulfurreducens
Authors: Gaspard Sarra , Vazquez Francisco , Holliger Christof 
Published: December 2019

ABSTRACT

The iron(III) reductase activity of
Geobacter sulfurreducens
was determined with the electron donor NADH and the artificial electron donor horse heart cytochrome
c
. The highest reduction rates were obtained with Fe(III) complexed by nitrilotriacetic acid as an electron acceptor. Fractionation experiments indicated that no iron(III) reductase activity was present in the cytoplasm, that approximately one-third was found in the periplasmic fraction, and that two-thirds were associated with the membrane fraction. Sucrose gradient separation of the outer and cytoplasmic membranes showed that about 80% of the iron(III) reductase was present in the outer membrane. The iron(III) reductase could be solubilized from the membrane fraction with 0.5 M KCl showing that the iron(III) reductase was weakly bound to the membranes. In addition, solubilization of the iron(III) reductase from whole cells with 0.5 M KCl, without disruption of cells, indicated that the iron(III) reductase is a peripheral protein on the outside of the outer membrane. Redox difference spectra of KCl extracts showed the presence of
c
-type cytochromes which could be oxidized by ferrihydrite. Only one activity band was observed in native polyacrylamide gels stained for the iron(III) reductase activity. Excision of the active band from a preparative gel followed by extraction of the proteins and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of high-molecular-mass, cytochrome-containing proteins in this iron(III) reductase activity band. From these experimental data it can be hypothesized that the iron(III) reductase of
G. sulfurreducens
is a peripheral outer membrane protein that might contain a
c
-type cytochrome.
Role of the Regulatory Gene
areA
of
Aspergillus oryzae
in Nitrogen Metabolism
Authors: Christensen Tove , Hynes Michael J. , Davis Meryl A. 
Published: December 2019

ABSTRACT

The
areA
gene of
Aspergillus oryzae
was cloned by cross-hybridization with the
Aspergillus nidulans areA
gene and was found to encode an 866-amino-acid protein that is very similar to other fungal nitrogen regulatory proteins. The
A. oryzae areA
gene can complement
A. nidulans areA
loss-of-function mutations. Functional analyses indicated that the N-terminal region of the
A. oryzae
AreA protein was dispensable for function and revealed a probable acidic activation domain in the protein. C-terminal truncation of the protein resulted in derepression of several nitrogen-controlled activities in
A. nidulans
, while deletions extending into the conserved GATA type zinc finger region abolished the activator function. The
A. oryzae areA
gene was inactivated by replacement with the
A. oryzae pyrG
gene. Strains containing the resulting
areA
deletion grew as well as the wild-type strain on glutamine but were unable to grow vigorously on other nitrogen sources, including ammonium. While
A. oryzae
exhibited reduced growth on 10 mM ammonium, the results of growth tests indicated that
areA
mutants of both
A. oryzae
and
A. nidulans
were affected in utilization of low concentrations of ammonium. The levels of the major nitrogen assimilatory enzymes, NADP-linked glutamate dehydrogenase (EC
1.4.1.4
) and glutamine synthetase (EC
6.3.1.2
), were determined. In both
A. oryzae
and
A. nidulans areA
mutants, the NADP-glutamate dehydrogenase levels were reduced, whereas the glutamine synthetase levels were not affected. These results suggest that the AreA protein may play an important role in the regulation of nitrogen assimilation in addition to its previously established regulatory role in nitrogen catabolism.
Nucleic Acid (DNA, RNA) Quantification and RNA/DNA Ratio Determination in Marine Sediments: Comparison of Spectrophotometric, Fluorometric, and HighPerformance Liquid Chromatography Methods and Estimation of Detrital DNA
Authors: Dell’Anno A. , Fabiano M. , Duineveld G. C. A. , Kok A. , Danovaro R. 
Published: December 2019

ABSTRACT
In this study, we compared three methods for extraction and quantification of RNA and DNA from marine sediments: (i) a spectrophotometric method using the diphenylamine assay; (ii) a fluorometric method utilizing selective fluorochromes (thiazole orange for total nucleic acids and Hoechst 33258 for DNA); and (iii) a high-pressure liquid chromatography (HPLC) method which uses a specific column to separate RNA and DNA and UV absorption of the nucleic acids for quantification. Sediment samples were collected in the oligotrophic Cretan Sea (eastern Mediterranean, from 40 to 1,540 m in depth) and compared to the distribution and composition of the benthic microbial assemblages (i.e., bacteria and microprotozoa). DNA concentrations measured spectrophotometrically and by HPLC were not significantly different, while fluorometric yields were significantly lower. Such differences appear mainly due to fact that the stain-DNA complex is strongly dependent on the DNA composition and structure. RNA concentrations determined by the three methods displayed some differences; fluorometric and spectrophotometric methods obtain RNA concentration by difference and therefore may be biased by DNA estimates. By contrast, the HPLC method provides independent assessments of RNA and DNA concentrations. We tentatively estimated the contribution of the detrital DNA to the total DNA pools in two ways. The two calculations provided quite similar results indicating that the majority of the DNA pool in the deep-sea sediments was detrital. Microbial RNA generally accounted for almost the entire sedimentary RNA pools below 100-m depth. RNA concentrations were found to decrease along the Cretan shelf and slope. The RNA/DNA ratio calculated by using fluorometric DNA concentrations was significantly correlated with values of sediment community oxygen consumption only below 100-m depth (dominated by the microbial biomass). These data suggest that the RNA/DNA ratio, based on fluorometric estimates of DNA, can be used as an indicator of benthic metabolic activity, but only when metazoan contribution to the microbial DNA is negligible.
Substrate Specificity of and Product Formation by Muconate Cycloisomerases: an Analysis of Wild-Type Enzymes and Engineered Variants
Authors: Vollmer Martin Dominik , Hoier Helga , Hecht Hans-Jürgen , Schell Ursula , Gröning Janosch , Goldman Adrian , Schlömann Michael 
Published: December 2019

ABSTRACT

Muconate cycloisomerases play a crucial role in the bacterial degradation of aromatic compounds by converting
cis,cis
-muconate, the product of catechol ring cleavage, to (4
S
)-muconolactone. Chloromuconate cycloisomerases catalyze both the corresponding reaction and a dehalogenation reaction in the transformation of chloroaromatic compounds. This study reports the first thorough examination of the substrate specificity of the muconate cycloisomerases from
Pseudomonas putida
PRS2000 and
Acinetobacter
“
calcoaceticus
” ADP1. We show that they transform, in addition to
cis,cis
-muconate, 3-fluoro-, 2-methyl-, and 3-methyl-
cis,cis
-muconate with high specificity constants but not 2-fluoro-, 2-chloro-, 3-chloro-, or 2,4-dichloro-
cis,cis
-muconate. Based on known three-dimensional structures, variants of
P. putida
muconate cycloisomerase were constructed by site-directed mutagenesis to contain amino acids found in equivalent positions in chloromuconate cycloisomerases. Some of the variants had significantly increased specificity constants for 3-chloro- or 2,4-dichloromuconate (e.g., A271S and I54V showed 27- and 22-fold increases, respectively, for the former substrate). These kinetic improvements were not accompanied by a change from protoanemonin to
cis,cis
-dienelactone as the product of 3-chloro-
cis,cis
-muconate conversion. The rate of 2-chloro-
cis,cis
-muconate turnover was not significantly improved, nor was this compound dehalogenated to any significant extent. However, the direction of 2-chloro-
cis,cis
-muconate cycloisomerization could be influenced by amino acid exchange. While the wild-type enzyme discriminated only slightly between the two possible cycloisomerization directions, some of the enzyme variants showed a strong preference for either (+)-2-chloro- or (+)-5-chloromuconolactone formation. These results show that the different catalytic characteristics of muconate and chloromuconate cycloisomerases are due to a number of features that can be changed independently of each other.
Purification and Characterization of
l
-Methionine γ-Lyase from
Brevibacterium linens
BL2
Authors: Dias Benjamin , Weimer Bart 
Published: December 2019

ABSTRACT

l
-Methionine γ-lyase (EC
4.4.1.11
) was purified to homogeneity from
Brevibacterium linens
BL2, a coryneform bacterium which has been used successfully as an adjunct bacterium to improve the flavor of Cheddar cheese. The enzyme catalyzes the α,γ elimination of methionine to produce methanethiol, α-ketobutyrate, and ammonia. It is a pyridoxal phosphate-dependent enzyme, with a native molecular mass of approximately 170 kDa, consisting of four identical subunits of 43 kDa each. The purified enzyme had optimum activity at pH 7.5 and was stable at pHs ranging from 6.0 to 8.0 for 24 h. The pure enzyme had its highest activity at 25°C but was active between 5 and 50°C. Activity was inhibited by carbonyl reagents, completely inactivated by
dl
-propargylglycine, and unaffected by metal-chelating agents. The pure enzyme had catalytic properties similar to those of
l
-methionine γ-lyase from
Pseudomonas putida
. Its

K
m

for the catalysis of methionine was 6.12 mM, and its maximum rate of catalysis was 7.0 μmol min
−1
mg
−1
. The enzyme was active under salt and pH conditions found in ripening Cheddar cheese but susceptible to degradation by intracellular proteases.
Growth Rate Regulation of rRNA Content of a Marine
Synechococcus
(Cyanobacterium) Strain
Authors: Binder Brian J. , Liu Ying Chun 
Published: December 2019

ABSTRACT

The relationship between growth rate and rRNA content in a marine
Synechococcus
strain was examined. A combination of flow cytometry and whole-cell hybridization with fluorescently labeled 16S rRNA-targeted oligonucleotide probes was used to measure the rRNA content of
Synechococcus
strain WH8101 cells grown at a range of light-limited growth rates. The sensitivity of this approach was sufficient for the analysis of rRNA even in very slowly growing
Synechococcus
cells (μ = 0.15 day
−1
). The relationship between growth rate and cellular rRNA content comprised three phases: (i) at low growth rates (<∼0.7 day
−1
), rRNA cell
−1
remained approximately constant; (ii) at intermediate rates (∼0.7 − 1.6 day
−1
), rRNA cell
−1
increased proportionally with growth rate; and (iii) at the highest, light-saturated rates (>∼1.6 day
−1
), rRNA cell
−1
dropped abruptly. Total cellular RNA (as measured with the nucleic acid stain SYBR Green II) was well correlated with the probe-based measure of rRNA and varied in a similar manner with growth rate. Mean cell volume and rRNA concentration (amount of rRNA per cubic micrometer) were related to growth rate in a manner similar to rRNA cell
−1
, although the overall magnitude of change in both cases was reduced. These patterns are hypothesized to reflect an approximately linear increase in ribosome efficiency with increasing growth rate, which is consistent with the prevailing prokaryotic model at low growth rates. Taken together, these results support the notion that measurements of cellular rRNA content might be useful for estimating in situ growth rates in natural
Synechococcus
populations.
Direct Determination of Carbon and Nitrogen Contents of Natural Bacterial Assemblages in Marine Environments
Authors: Fukuda Rumi , Ogawa Hiroshi , Nagata Toshi , Koike Isao 
Published: December 2019

ABSTRACT

In order to better estimate bacterial biomass in marine environments, we developed a novel technique for direct measurement of carbon and nitrogen contents of natural bacterial assemblages. Bacterial cells were separated from phytoplankton and detritus with glass fiber and membrane filters (pore size, 0.8 μm) and then concentrated by tangential flow filtration. The concentrate was used for the determination of amounts of organic carbon and nitrogen by a high-temperature catalytic oxidation method, and after it was stained with 4′,6-diamidino-2-phenylindole, cell abundance was determined by epifluorescence microscopy. We found that the average contents of carbon and nitrogen for oceanic bacterial assemblages were 12.4 ± 6.3 and 2.1 ± 1.1 fg cell
−1
(mean ± standard deviation;
n
= 6), respectively. Corresponding values for coastal bacterial assemblages were 30.2 ± 12.3 fg of C cell
−1
and 5.8 ± 1.5 fg of N cell
−1
(
n
= 5), significantly higher than those for oceanic bacteria (two-tailed Student’s
t
test;
P
< 0.03). There was no significant difference (
P
> 0.2) in the bacterial C:N ratio (atom atom
−1
) between oceanic (6.8 ± 1.2) and coastal (5.9 ± 1.1) assemblages. Our estimates support the previous proposition that bacteria contribute substantially to total biomass in marine environments, but they also suggest that the use of a single conversion factor for diverse marine environments can lead to large errors in assessing the role of bacteria in food webs and biogeochemical cycles. The use of a factor, 20 fg of C cell
−1
, which has been widely adopted in recent studies may result in the overestimation (by as much as 330%) of bacterial biomass in open oceans and in the underestimation (by as much as 40%) of bacterial biomass in coastal environments.
Identification and Activities In Situ of
Nitrosospira
and
Nitrospira
spp. as Dominant Populations in a Nitrifying Fluidized Bed Reactor
Authors: Schramm Andreas , de Beer Dirk , Wagner Michael , Amann Rudolf 
Published: December 2019

ABSTRACT

Bacterial aggregates from a chemolithoautotrophic, nitrifying fluidized bed reactor were investigated with microsensors and rRNA-based molecular techniques. The microprofiles of O
2
, NH
4
+
, NO
2
−
, and NO
3
−
demonstrated the occurrence of complete nitrification in the outer 125 μm of the aggregates. The ammonia oxidizers were identified as members of the
Nitrosospira
group by fluorescence in situ hybridization (FISH). No ammonia- or nitrite-oxidizing bacteria of the genus
Nitrosomonas
or
Nitrobacter
, respectively, could be detected by FISH. To identify the nitrite oxidizers, a 16S ribosomal DNA clone library was constructed and screened by denaturing gradient gel electrophoresis and selected clones were sequenced. The organisms represented by these sequences formed two phylogenetically distinct clusters affiliated with the nitrite oxidizer
Nitrospira moscoviensis
. 16S rRNA-targeted oligonucleotide probes were designed for in situ detection of these organisms. FISH analysis showed that the dominant populations of
Nitrospira
spp. and
Nitrosospira
spp. formed separate, dense clusters which were in contact with each other and occurred throughout the aggregate. A second, smaller, morphologically and genetically different population of
Nitrospira
spp. was restricted to the outer nitrifying zones.
Microbial Desulfurization of a Crude Oil Middle-Distillate Fraction: Analysis of the Extent of Sulfur Removal and the Effect of Removal on Remaining Sulfur
Authors: Grossman M. J. , Lee M. K. , Prince R. C. , Garrett K. K. , George G. N. , Pickering I. J. 
Published: December 2019

ABSTRACT

Rhodococcus
sp. strain ECRD-1 was evaluated for its ability to desulfurize a 232 to 343°C middle-distillate (diesel range) fraction of Oregon basin (OB) crude oil. OB oil was provided as the sole source of sulfur in batch cultures, and the extent of desulfurization and the chemical fate of the residual sulfur in the oil after treatment were determined. Gas chromatography (GC), flame ionization detection, and GC sulfur chemiluminesce detection analysis were used to qualitatively evaluate the effect of
Rhodococcus
sp. strain ECRD-1 treatment on the hydrocarbon and sulfur content of the oil, respectively. Total sulfur was determined by combustion of samples and measurement of released sulfur dioxide by infrared absorption. Up to 30% of the total sulfur in the middle distillate cut was removed, and compounds across the entire boiling range of the oil were affected. Sulfur K-edge X-ray absorption-edge spectroscopy was used to examine the chemical state of the sulfur remaining in the treated OB oil. Approximately equal amounts of thiophenic and sulfidic sulfur compounds were removed by ECRD-1 treatment, and over 50% of the sulfur remaining after treatment was in an oxidized form. The presence of partially oxidized sulfur compounds indicates that these compounds were en route to desulfurization. Overall, more than two-thirds of the sulfur had been removed or oxidized by the microbial treatment.