bluuebunny/crossref_metadata_2025 huggingface dataset:
Formation of Bound Residues during Microbial Degradation of [
14
C]Anthracene in Soil
Authors: Kästner M. , Streibich S. , Beyrer M. , Richnow H. H. , Fritsche W.
Published: December 2019

Carbon partitioning and residue formation during microbial degradation of polycyclic aromatic hydrocarbons (PAH) in soil and soil-compost mixtures were examined by using [
14
C]anthracenes labeled at different positions. In native soil 43.8% of [9-
14
C]anthracene was mineralized by the autochthonous microflora and 45.4% was transformed into bound residues within 176 days. Addition of compost increased the metabolism (67.2% of the anthracene was mineralized) and decreased the residue formation (20.7% of the anthracene was transformed). Thus, the higher organic carbon content after compost was added did not increase the level of residue formation. [
14
C]anthracene labeled at position 1,2,3,4,4a,5a was metabolized more rapidly and resulted in formation of higher levels of residues (28.5%) by the soil-compost mixture than [
14
C]anthracene radiolabeled at position C-9 (20.7%). Two phases of residue formation were observed in the experiments. In the first phase the original compound was sequestered in the soil, as indicated by its limited extractability. In the second phase metabolites were incorporated into humic substances after microbial degradation of the PAH (biogenic residue formation). PAH metabolites undergo oxidative coupling to phenolic compounds to form nonhydrolyzable humic substance-like macromolecules. We found indications that monomeric educts are coupled by C-C- or either bonds. Hydrolyzable ester bonds or sorption of the parent compounds plays a minor role in residue formation. Moreover, experiments performed with
14
CO
2
revealed that residues may arise from CO
2
in the soil in amounts typical for anthracene biodegradation. The extent of residue formation depends on the metabolic capacity of the soil microflora and the characteristics of the soil. The position of the
14
C label is another important factor which controls mineralization and residue formation from metabolized compounds.
Export of Virulence Genes and Shiga Toxin by Membrane Vesicles of
Escherichia coli
O157:H7
Authors: Kolling Glynis L. , Matthews Karl R.
Published: December 2019

Membrane vesicles released by
Escherichia coli
O157:H7 into culture medium were purified and analyzed for protein and DNA content. Electron micrographs revealed vesicles that are spherical, range in size from 20 to 100 nm, and have a complete bilayer. Analysis of vesicle protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates vesicles that contain many proteins with molecular sizes similar to outer membrane proteins and a number of cellular proteins. Immunoblot (Western) analysis of vesicles suggests the presence of cell antigens. Treatment of vesicles with exogenous DNase hydrolyzed surface-associated DNA; PCR demonstrated that vesicles contain DNA encoding the virulence genes
eae
,
stx1
and
stx2
, and
uidA
, which encodes for β-galactosidase. Immunoblot analysis of intact and lysed, proteinase K-treated vesicles demonstrate that Shiga toxins 1 and 2 are contained within vesicles. These results suggest that vesicles contain toxic material and transfer experiments demonstrate that vesicles can deliver genetic material to other gram-negative organisms.
A Colorimetric Technique for Detecting Trichothecenes and Assessing Relative Potencies
Authors: Engler Kathryn H. , Coker Raymond D. , Evans Ivor H.
Published: December 2019

We tested a novel colorimetric toxicity test, based on inhibition of β-galactosidase activity in the yeast
Kluyveromyces marxianus
, for sensitivity to a range of mycotoxins. A variety of trichothecene mycotoxins could be detected. The order of toxicity established with this bioassay was verrucarin A > roridin A > T-2 toxin > diacetoxyscirpenol > HT-2 toxin > acetyl T-2 toxin > neosolaniol > fusarenon X > T-2 triol > scirpentriol > nivalenol > deoxynivalenol > T-2 tetraol. The sensitivity of detection was high, with the most potent trichothecene tested, verrucarin A, having a 50% effective concentration (concentration of toxin causing 50% inhibition) of 2 ng/ml. Other mycotoxins (cyclopiazonic acid, fumonisin B
1
, ochratoxin A, patulin, sterigmatocystin, tenuazonic acid, and zearalenone) could not be detected at up to 10 μg/ml, nor could aflatoxins B
1
and M
1
be detected at concentrations up to 25 μg/ml. This test should be useful for trichothecene detection and for studies of relevant interactions—both between trichothecenes themselves and between trichothecenes and other food constituents.
Differentiation of
Vibrio alginolyticus
Strains Isolated from Sardinian Waters by Ribotyping and a New Rapid PCR Fingerprinting Method
Authors: Zanetti Stefania , Deriu Antonella , Duprè Ilaria , Sanguinetti Maurizio , Fadda Giovanni , Sechi Leonardo A.
Published: December 2019

We investigated the usefulness of a novel PCR fingerprinting technique, based on the specific amplification of genomic regions, to differentiate 30
Vibrio alginolyticus
strains isolated in Sardinian waters. The different profiles obtained were scanned and analyzed by a computer program in order to determine genetic relationships. The results were then compared with the patterns obtained by ribotyping with
Hin
dIII,
Kpn
I, and
Xba
I restriction enzymes. PCR fingerprinting could differentiate the strains analyzed into 12 different patterns, whereas ribotyping with
Xba
I, which produced the highest number of patterns, generated only 7 different profiles. This study revealed the superior discriminative power of the proposed technique for the differentiation of related
V. alginolyticus
strains and the potential use of PCR fingerprinting in epidemiological studies.
Viral Lysis and Bacterivory during a Phytoplankton Bloom in a Coastal Water Microcosm
Authors: Guixa-Boixereu Núria , Lysnes Kristine , Pedrós-Alió Carlos
Published: December 2019

The relative importance of viral lysis and bacterivory as causes of bacterial mortality were estimated. A laboratory experiment was carried out to check the kind of control that viruses could exert over the bacterial assemblage in a non-steady-state situation. Virus-like particles (VLP) were determined by using three methods of counting (DAPI [4′,6-diamidino-2-phenylindole] staining, YOPRO staining, and transmission electron microscopy). Virus counts increased from the beginning until the end of the experiment. However, different methods produced significantly different results. DAPI-stained VLP yielded the lowest numbers, while YOPRO-stained VLP yielded the highest numbers. Bacteria reached the maximal abundance at 122 h (3 × 10
7
bacteria ml
−1
), after the peak of chlorophyll
a
(80 μg liter
−1
). Phototrophic nanoflagellates followed the same pattern as for chlorophyll
a
. Heterotrophic nanoflagellates showed oscillations in abundance throughout the experiment. The specific bacterial growth rate increased until 168 h (2.6 day
−1
). The bacterivory rate reached the maximal value at 96 hours (0.9 day
−1
). Bacterial mortality due to viral infection was measured by using two approaches: measuring the percentage of visibly infected bacteria (%VIB) and measuring the viral decay rates (VDR), which were estimated with cyanide. The %VIB was always lower than 1% during the experiment. VDR were used to estimate viral production. Viral production increased 1 order of magnitude during the experiment (from 10
6
to 10
7
VLP ml
−1
h
−1
). The percentage of heterotrophic bacterial production consumed by bacterivores was higher than 60% during the first 4 days of the experiment; afterwards, this percentage was lower than 10%. The percentage of heterotrophic bacterial production lysed by viruses as assessed by the VDR reached the highest values at the beginning (100%) and at the end (50%) of the experiment. Comparing both sources of mortality at each stage of the bloom, bacterivory was found to be higher than viral lysis at days 2 and 4, and viral lysis was higher than bacterivory at days 7 and 9. A balance between bacterial losses and bacterial production was calculated for each sampling interval. At intervals of 0 to 2 and 2 to 4 days, viral lysis and bacterivory accounted for all the bacterial losses. At intervals of 4 to 7 and 7 to 9 days, bacterial losses were not balanced by the sources of mortality measured. At these time points, bacterial abundance was about 20 times higher than the expected value if viral lysis and bacterivory had been the only factors causing bacterial mortality. In conclusion, mortality caused by viruses can be more important than bacterivory under non-steady-state conditions.
Purification and Characterization of an Extremely Thermostable Cyclomaltodextrin Glucanotransferase from a Newly Isolated Hyperthermophilic Archaeon, a
Thermococcus
sp
Authors: Tachibana Yoshihisa , Kuramura Akiko , Shirasaka Naoki , Suzuki Yuji , Yamamoto Tomoko , Fujiwara Shinsuke , Takagi Masahiro , Imanaka Tadayuki
Published: December 2019

The extremely thermophilic anaerobic archaeon strain B1001 was isolated from a hot-spring environment in Japan. The cells were irregular cocci, 0.5 to 1.0 μm in diameter. The new isolate grew at temperatures between 60 and 95°C (optimum, 85°C), from pH 5.0 to 9.0 (optimum, pH 7.0), and from 1.0 to 6.0% NaCl (optimum, 2.0%). The G+C content of the genomic DNA was 43.0 mol%. The 16S rRNA gene sequencing of strain B1001 indicated that it belongs to the genus
Thermococcus
. During growth on starch, the strain produced a thermostable cyclomaltodextrin glucanotransferase (CGTase). The enzyme was purified 1,750-fold, and the molecular mass was determined to be 83 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Incubation at 120°C with SDS and 2-mercaptoethanol was required for complete unfolding. The optimum temperatures for starch-degrading activity and cyclodextrin synthesis activity were 110 and 90 to 100°C, respectively. The optimum pH for enzyme activity was pH 5.0 to 5.5. At pH 5.0, the half-life of the enzyme was 40 min at 110°C. The enzyme formed mainly α-cyclodextrin with small amounts of β- and γ-cyclodextrins from starch. This is the first report on the presence of the extremely thermostable CGTase from hyperthermophilic archaea.
Structure and Dynamics of Experimentally Introduced and Naturally Occurring
Laccaria
sp. Discrete Genotypes in a Douglas Fir Plantation
Authors: Selosse Marc-André , Martin Francis , Bouchard Daniel , le Tacon François
Published: December 2019

Ectomycorrhizal fungi have been introduced in forest nurseries to improve seedling growth. Outplanting of inoculated seedlings to forest plantations raises the questions about inoculant persistence and its effects on indigenous fungal populations. We previously showed (M.-A. Selosse et al. Mol. Ecol. 7:561–573, 1998) that the American strain
Laccaria bicolor
S238N persisted 10 years after outplanting in a French Douglas fir plantation, without introgression or selfing and without fruiting on uninoculated adjacent plots. In the present study, the relevance of those results to sympatric strains was assessed for another part of the plantation, planted in 1985 with seedlings inoculated with the French strain
L. bicolor
81306 or left uninoculated. About 720
Laccaria
sp. sporophores, collected from 1994 to 1997, were typed by using randomly amplified polymorphic DNA markers and PCR amplification of the mitochondrial and nuclear ribosomal DNAs. All plots were colonized by small spontaneous discrete genotypes (genets). The inoculant strain 81306 abundantly fruited beneath inoculated trees, with possible introgression in indigenous
Laccaria
populations but without selfing. In contrast to our previous survey of
L. bicolor
S238N,
L. bicolor
81306 colonized a plot of uninoculated trees. Meiotic segregation analysis verified that the invading genet was strain 81306 (
P
< 0.00058), implying a vegetative growth of 1.1 m · year
−1
. This plot was also invaded in 1998 by strain S238N used to inoculate other trees of the plantation. Five other uninoculated plots were free of these inoculant strains. The fate of inoculant strains thus depends less on their geographic origin than on unknown local factors.
Enhanced Nitrogen Fixation in a
Rhizobium etli ntrC
Mutant That Overproduces the
Bradyrhizobium japonicum
Symbiotic Terminal Oxidase
cbb
3
Authors: Soberón Mario , López Oswaldo , Morera Claudia , Girard Maria de Lourdes , Tabche Maria Luisa , Miranda Juan
Published: December 2019

The
ntrC
gene codes for a transcriptional activator protein that modulates gene expression in response to nitrogen. The cytochrome production pattern of a
Rhizobium etli ntrC
mutant (CFN2012) was studied. CO difference spectral analysis of membranes showed that CFN2012 produced a terminal oxidase similar to the symbiotic terminal oxidase of bacteroids in free-living cells under aerobic conditions, with a characteristic trough at 553 nm. CFN2012 produced two
c
-type cytochromes with molecular masses of 27 and 32 kDa, in contrast with the wild-type strain, which produced only a 32-kDa
c
-type cytochrome. The expression levels of the
R. etli fixNOQP
operon, which codes for terminal oxidase
cbb
3
, were not affected by the
ntrC
mutation. However, the production levels of the two
c
-type cytochromes (27 and 32 kDa) were enhanced at least eightfold when the
Bradyrhizobium japonicum fixNOQP
operon was expressed in CFN2012 from the
nptII
promoter (pMSfix
c
), suggesting that these proteins are subunits FixO (27 kDa) and FixP (32 kDa) of
cbb
3
and that CFN2012/pMSfix
c
overproduced this terminal oxidase. CFN2012/pMSfix
c
showed a significant increase in its symbiotic performance as judged by the determination of nitrogenase activities of plants inoculated with this strain, suggesting that the overproduction of
cbb
3
terminal oxidase correlates with an enhancement in symbiotic nitrogen fixation.
Cloning and Characterization of Polyphosphate Kinase and Exopolyphosphatase Genes from
Pseudomonas aeruginosa
8830
Authors: Zago Anna , Chugani Sudha , Chakrabarty A. M.
Published: December 2019

Pseudomonas aeruginosa
accumulates polyphosphates in response to nutrient limitations. To elucidate the function of polyphosphate in this microorganism, we have investigated polyphosphate metabolism by isolating from
P. aeruginosa
8830 the genes encoding polyphosphate kinase (PPK) and exopolyphosphatase (PPX), which are involved in polyphosphate synthesis and degradation, respectively. The 690- and 506-amino-acid polypeptides encoded by the two genes have been expressed in
Escherichia coli
and purified, and their activities have been tested in vitro. Gene replacement was used to construct a PPK-negative strain of
P. aeruginosa
8830. Low residual PPK activity in the
ppk
mutant suggests a possible alternative pathway of polyphosphate synthesis in this microorganism. Primer extension analysis indicated that
ppk
is transcribed from a ς
E
-dependent promoter, which could be responsive to environmental stresses. However, no coregulation between
ppk
and
ppx
promoters has been demonstrated in response to osmotic shock or oxidative stress.
Lignin-Modifying Enzymes of
Flavodon flavus
, a Basidiomycete Isolated from a Coastal Marine Environment
Authors: Raghukumar C. , D’Souza T. M. , Thorn R. G. , Reddy C. A.
Published: December 2019

A basidiomycetous fungus
Flavodon flavus
(Klotzsch) Ryvarden (strain 312), isolated from decaying sea grass from a coral lagoon off the west coast of India, mineralized nearly 24% of
14
C-labeled synthetic lignin to
14
CO
2
in 24 days. When grown in low-nitrogen medium (2.4 mM N) this fungus produced three major classes of extracellular lignin-modifying enzymes (LMEs): manganese-dependent peroxidase (MNP), lignin peroxidase (LIP), and laccase. Low MNP and laccase activities were seen in high-nitrogen medium (24 mM N), but no LIP activity was seen. In media containing lignocellulosic substrates such as pine, poplar, or sugarcane bagasse as the sole source of carbon and nitrogen, relatively high MNP and moderate levels of laccases were seen, but LIP production either was not seen or was minimal. LME production was also seen in media prepared with artificial seawater. Fast protein liquid chromatography and isoelectric focusing resolved LMEs into four isozymes each of MNP and LIP, while laccase isozymes were resolved into two groups, one group containing seven isozymes (pIs 4 to 6) and the other group containing at least three isozymes (pIs < 3). The molecular masses of the different isozymes were 43 to 99 kDa for MNP, 40 and 41.5 kDa for LIP, and 43 and 99 kDa for laccase.
F. flavus
showed effective degradation of various dye pollutants in media prepared with or without artificial seawater. This is the first report on the production of all three major classes of LMEs by
F. flavus
and points to the bioremediation potential of this organism in terrestrial as well as marine environments.
Detection of Viable
Listeria monocytogenes
with a 5′ Nuclease PCR Assay
Authors: Norton Dawn-Marie , Batt Carl A.
Published: December 2019

A 5′ nuclease assay has been developed to detect viable
Listeria monocytogenes
. The assay targets the hemolysin A (
hlyA
) transcript, which is found only in
L. monocytogenes
. The single-tube, reverse transcriptase (RT), fluorogenic probe-based assay was formatted by using
Tth
DNA polymerase whose activity was modulated by using the manganese-chelating morpholinepropanesulfonic acid (MOPS) buffer. This assay was quantitative over a 3-log-unit range of template concentrations when tested with an in vitro-transcribed
hlyA
mRNA. The viability of
L. monocytogenes
was reduced by heating at various temperatures and times up to a maximum of a 9-D inactivation. The location of the primer had a pronounced effect on the utility of the assay, and primers located in the most distal regions of the
hlyA
transcript appeared to correlate with the number of CFU while primers located more internal on the amplicon overestimated the cell viability. The assay with primers that included the 3′ end of the transcript was an accurate indicator of viability as measured by CFU determination or staining with 5-sulfofluorescein diacetate.
Growth from Spores of NonproteolyticClostridium botulinumin Heat-Treated Vegetable Juice
Authors: Stringer Sandra C. , Haque Nuzrul , Peck Michael W.
Published: December 2019

Unheated spores of nonproteolyticClostridium botulinumwere able to lead to growth in sterile deoxygenated turnip, spring green, helda bean, broccoli, or potato juice, although the probability of growth was low and the time to growth was longer than the time to growth in culture media. With all five vegetable juices tested, the probability of growth increased when spores were inoculated into the juice and then heated for 2 min in a water bath at 80°C. The probability of growth was greater in bean or broccoli juice than in culture media following 10 min of heat treatment in these media. Growth was prevented by heat treatment of spores in vegetable juices or culture media at 80°C for 100 min. We show for the first time that adding heat-treated vegetable juice to culture media can increase the number of heat-damaged spores ofC. botulinumthat can lead to colony formation.
Cloning, Expression, and Nucleotide Sequence of the
Pseudomonas aeruginosa
142
ohb
Genes Coding for Oxygenolytic
ortho
Dehalogenation of Halobenzoates
Authors: Tsoi Tamara V. , Plotnikova Elena G. , Cole James R. , Guerin William F. , Bagdasarian Michael , Tiedje James M.
Published: December 2019

We have cloned and characterized novel oxygenolytic
ortho
-dehalogenation (
ohb
) genes from 2-chlorobenzoate (2-CBA)- and 2,4-dichlorobenzoate (2,4-dCBA)-degrading
Pseudomonas aeruginosa
142. Among 3,700
Escherichia coli
recombinants, two clones, DH5αF′(pOD22) and DH5αF′(pOD33), converted 2-CBA to catechol and 2,4-dCBA and 2,5-dCBA to 4-chlorocatechol. A subclone of pOD33, plasmid pE43, containing the 3,687-bp minimized
ohb
DNA region conferred to
P. putida
PB2440 the ability to grow on 2-CBA as a sole carbon source. Strain PB2440(pE43) also oxidized but did not grow on 2,4-dCBA, 2,5-dCBA, or 2,6-dCBA. Terminal oxidoreductase ISP
OHB
structural genes
ohbA
and
ohbB
, which encode polypeptides with molecular masses of 20,253 Da (β-ISP) and 48,243 Da (α-ISP), respectively, were identified; these proteins are in accord with the 22- and 48-kDa (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) polypeptides synthesized in
E. coli
and
P. aeruginosa
parental strain 142. The
ortho
-halobenzoate 1,2-dioxygenase activity was manifested in the absence of ferredoxin and reductase genes, suggesting that the ISP
OHB
utilized electron transfer components provided by the heterologous hosts. ISP
OHB
formed a new phylogenetic cluster that includes aromatic oxygenases featuring atypical structural-functional organization and is distant from the other members of the family of primary aromatic oxygenases. A putative IclR-type regulatory gene (
ohbR
) was located upstream of the
ohbAB
genes. An open reading frame (
ohbC
) of unknown function that overlaps lengthwise with
ohbB
but is transcribed in the opposite direction was found. The
ohbC
gene codes for a 48,969-Da polypeptide, in accord with the 49-kDa protein detected in
E. coli
. The
ohb
genes are flanked by an IS
1396
-like sequence containing a putative gene for a 39,715-Da transposase A (
tnpA
) at positions 4731 to 5747 and a putative gene for a 45,247-Da DNA topoisomerase I/III (
top
) at positions 346 to 1563. The
ohb
DNA region is bordered by 14-bp imperfect inverted repeats at positions 56 to 69 and 5984 to 5997.
Molecular Phylogenetic and Biogeochemical Studies of Sulfate-Reducing Bacteria in the Rhizosphere of
Spartina alterniflora
Authors: Hines Mark E. , Evans Robert S. , Sharak Genthner Barbara R. , Willis Stephanie G. , Friedman Stephanie , Rooney-Varga Juliette N. , Devereux Richard
Published: December 2019

The population composition and biogeochemistry of sulfate-reducing bacteria (SRB) in the rhizosphere of the marsh grass
Spartina alterniflora
was investigated over two growing seasons by molecular probing, enumerations of culturable SRB, and measurements of SO
4
2−
reduction rates and geochemical parameters. SO
4
2−
reduction was rapid in marsh sediments with rates up to 3.5 μmol ml
−1
day
−1
. Rates increased greatly when plant growth began in April and decreased again when plants flowered in late July. Results with nucleic acid probes revealed that SRB rRNA accounted for up to 43% of the rRNA from members of the domain
Bacteria
in marsh sediments, with the highest percentages occurring in bacteria physically associated with root surfaces. The relative abundance (RA) of SRB rRNA in whole-sediment samples compared to that of
Bacteria
rRNA did not vary greatly throughout the year, despite large temporal changes in SO
4
2−
reduction activity. However, the RA of root-associated SRB did increase from <10 to >30% when plants were actively growing. rRNA from members of the family
Desulfobacteriaceae
comprised the majority of the SRB rRNA at 3 to 34% of
Bacteria
rRNA, with
Desulfobulbus
spp. accounting for 1 to 16%. The RA of
Desulfovibrio
rRNA generally comprised from <1 to 3% of the
Bacteria
rRNA. The highest
Desulfobacteriaceae
RA in whole sediments was 26% and was found in the deepest sediment samples (6 to 8 cm). Culturable SRB abundance, determined by most-probable-number analyses, was high at >10
7
ml
−1
. Ethanol utilizers were most abundant, followed by acetate utilizers. The high numbers of culturable SRB and the high RA of SRB rRNA compared to that of
Bacteria
rRNA may be due to the release of SRB substrates in plant root exudates, creating a microbial food web that circumvents fermentation.
Removal of Dibenzofuran, Dibenzo-
p
-Dioxin, and 2-Chlorodibenzo-
p
-Dioxin from Soils Inoculated with
Sphingomonas
sp. Strain RW1
Authors: Halden Rolf U. , Halden Barbara G. , Dwyer Daryl F.
Published: December 2019

Removal of dibenzofuran, dibenzo-
p
-dioxin, and 2-chlorodibenzo-
p
-dioxin (2-CDD) (10 ppm each) from soil microcosms to final concentrations in the parts-per-billion range was affected by the addition of
Sphingomonas
sp. strain RW1. Rates and extents of removal were influenced by the density of RW1 organisms. For 2-CDD, the rate of removal was dependent on the content of soil organic matter (SOM), with half-life values ranging from 5.8 h (0% SOM) to 26.3 h (5.5% SOM).
Rapid Microbial Production of Filamentous Sulfur Mats at Hydrothermal Vents
Authors: Taylor Craig D. , Wirsen Carl O. , Gaill Françoise
Published: December 2019

During recent oceanographic cruises to Pacific hydrothermal vent sites (9°N and the Guaymas Basin), the rapid microbial formation of filamentous sulfur mats by a new chemoautotrophic, hydrogen sulfide-oxidizing bacterium was documented in both in situ and shipboard experiments. Observations suggest that formation of these sulfur mats may be a factor in the initial colonization of hydrothermal surfaces by macrofaunal
Alvinella
worms. This novel metabolic capability, previously shown to be carried out by a coastal strain in H
2
S continuous-flow reactors, may be an important, heretofore unconsidered, source of microbial organic matter production at deep-sea hydrothermal vents.
Elevated Growth of
Saccharomyces cerevisiae ATH1
Null Mutants on Glucose Is an Artifact of Nonmatching Auxotrophies of Mutant and Reference Strains
Authors: Chopra Rohini , Sharma Vishva Mitra , Ganesan K.
Published: December 2019

Yeast strains disrupted for
ATH1
, which encodes vacuolar acid trehalase, have been reported to grow to higher cell densities than reference strains. We showed that the increase in cell density is due to the
URA3
gene introduced as a part of the disruption and concluded that the misinterpretation is a result of not using a control strain with matching auxotrophic markers.
Stability of Related Human and Chicken
Campylobacter jejuni
Genotypes after Passage through Chick Intestine Studied by Pulsed-Field Gel Electrophoresis
Authors: Hänninen Marja-Liisa , Hakkinen Marjaana , Rautelin Hilpi
Published: December 2019

The genomic stability of 12
Campylobacter jejuni
strains consisting of two groups of human and chicken isolates was studied by analysis of their PFGE (pulsed-field gel electrophoresis) patterns after passage through newly hatched chicks’ intestines. The patterns of
Sma
I,
Sal
I, and
Sac
II digests remained stable after intestinal passage, except for those of two strains. One originally human strain, FB 6371, changed its genotype from II/A (
Sma
I/
Sac
II) to I/B. Another strain, BTI, originally isolated from a chicken, changed its genotype from I/B to a new genotype. The genomic instability of the strains was further confirmed by
Sal
I digestion and ribotyping of the
Hae
III digests. In addition, heat-stable serotype 57 of strain FB 6371 changed to serotype 27 in all isolates with new genotypes but remained unchanged in an isolate with the original genotype. Serotype 27 of strain BTI remained stable. Our study suggests that during intestinal colonization, genomic rearrangement, as demonstrated by changed PFGE and ribopatterns, may occur.
Characterization of Two Subsurface H
2
-Utilizing Bacteria,
Desulfomicrobium hypogeium
sp. nov. and
Acetobacterium psammolithicum
sp. nov., and Their Ecological Roles
Authors: Krumholz Lee R. , Harris Steve H. , Tay Stephen T. , Suflita Joseph M.
Published: December 2019

We examined the relative roles of acetogenic and sulfate-reducing bacteria in H
2
consumption in a previously characterized subsurface sandstone ecosystem. Enrichment cultures originally inoculated with ground sandstone material obtained from a Cretaceous formation in central New Mexico were grown with hydrogen in a mineral medium supplemented with 0.02% yeast extract. Sulfate reduction and acetogenesis occurred in these cultures, and the two most abundant organisms carrying out the reactions were isolated. Based on 16S rRNA analysis data and on substrate utilization patterns, these organisms were named
Desulfomicrobium hypogeium
sp. nov. and
Acetobacterium psammolithicum
sp. nov. The steady-state H
2
concentrations measured in sandstone-sediment slurries (threshold concentration, 5 nM), in pure cultures of sulfate reducers (threshold concentration, 2 nM), and in pure cultures of acetogens (threshold concentrations 195 to 414 nM) suggest that sulfate reduction is the dominant terminal electron-accepting process in the ecosystem examined. In an experiment in which direct competition for H
2
between
D. hypogeium
and
A. psammolithicum
was examined, sulfate reduction was the dominant process.
Reductive Dechlorination of Tetrachloroethene to
cis
-1,2-Dichloroethene by a Thermophilic Anaerobic Enrichment Culture
Authors: Kengen Servé W. M. , Breidenbach Caroline G. , Felske Andreas , Stams Alfons J. M. , Schraa Gosse , de Vos Willem M.
Published: December 2019

Thermophilic anaerobic biodegradation of tetrachloroethene (PCE) was investigated with various inocula from geothermal and nongeothermal areas. Only polluted harbor sediment resulted in a stable enrichment culture that converted PCE via trichloroethene to
cis
-1,2-dichloroethene at the optimum temperature of 60 to 65°C. After several transfers, methanogens were eliminated from the culture. Dechlorination was supported by lactate, pyruvate, fructose, fumarate, and malate as electron donor but not by H
2
, formate, or acetate. Fumarate and
l
-malate led to the highest dechlorination rate. In the absence of PCE, fumarate was fermented to acetate, H
2
, CO
2
, and succinate. With PCE, less H
2
was formed, suggesting that PCE competed for the reducing equivalents leading to H
2
. PCE dechlorination, apparently, was not outcompeted by fumarate as electron acceptor. At the optimum dissolved PCE concentration of ∼60 μM, a high dechlorination rate of 1.1 μmol h
−1
mg
−1
(dry weight) was found, which indicates that the dechlorination is not a cometabolic activity. Microscopic analysis of the fumarate-grown culture showed the dominance of a long thin rod. Molecular analysis, however, indicated the presence of two dominant species, both belonging to the low-G+C gram positives. The highest similarity was found with the genus
Dehalobacter
(90%), represented by the halorespiring organism
Dehalobacter restrictus
, and with the genus
Desulfotomaculum
(86%).
Chemoselective Nitro Group Reduction and Reductive Dechlorination Initiate Degradation of 2-Chloro-5-Nitrophenol by
Ralstonia eutropha
JMP134
Authors: Schenzle Andreas , Lenke Hiltrud , Spain Jim C. , Knackmuss Hans-Joachim
Published: December 2019

Ralstonia eutropha
JMP134 utilizes 2-chloro-5-nitrophenol as a sole source of nitrogen, carbon, and energy. The initial steps for degradation of 2-chloro-5-nitrophenol are analogous to those of 3-nitrophenol degradation in
R. eutropha
JMP134. 2-Chloro-5-nitrophenol is initially reduced to 2-chloro-5-hydroxylaminophenol, which is subject to an enzymatic Bamberger rearrangement yielding 2-amino-5-chlorohydroquinone. The chlorine of 2-amino-5-chlorohydroquinone is removed by a reductive mechanism, and aminohydroquinone is formed. 2-Chloro-5-nitrophenol and 3-nitrophenol induce the expression of 3-nitrophenol nitroreductase, of 3-hydroxylaminophenol mutase, and of the dechlorinating activity. 3-Nitrophenol nitroreductase catalyzes chemoselective reduction of aromatic nitro groups to hydroxylamino groups in the presence of NADPH. 3-Nitrophenol nitroreductase is active with a variety of mono-, di-, and trinitroaromatic compounds, demonstrating a relaxed substrate specificity of the enzyme. Nitrosobenzene serves as a substrate for the enzyme and is converted faster than nitrobenzene.
A Pyruvated Mannose-Specific Xanthan Lyase Involved in Xanthan Degradation by
Paenibacillus alginolyticus
XL-1
Authors: Ruijssenaars Harald J. , de Bont Jan A. M. , Hartmans Sybe
Published: December 2019

The xanthan-degrading bacterium
Paenibacillus alginolyticus
XL-1, isolated from soil, degrades approximately 28% of the xanthan molecule and appears to leave the backbone intact. Several xanthan-degrading enzymes were excreted during growth on xanthan, including xanthan lyase. Xanthan lyase production was induced by xanthan and inhibited by glucose and low-molecular-weight enzymatic degradation products from xanthan. A xanthan lyase with a molecular mass of 85 kDa and a pI of 7.9 was purified and characterized. The enzyme is specific for pyruvated mannosyl side chain residues and optimally active at pH 6.0 and 55°C.
Aggregation of
Bacillus thuringiensis
Cry1A Toxins upon Binding to Target Insect Larval Midgut Vesicles
Authors: Aronson Arthur I. , Geng Chaoxian , Wu Lan
Published: December 2019

During sporulation,
Bacillus thuringiensis
produces crystalline inclusions comprised of a mixture of δ-endotoxins. Following ingestion by insect larvae, these inclusion proteins are solubilized, and the protoxins are converted to toxins. These bind specifically to receptors on the surfaces of midgut apical cells and are then incorporated into the membrane to form ion channels. The steps required for toxin insertion into the membrane and possible oligomerization to form a channel have been examined. When bound to vesicles from the midguts of
Manduca sexta
larvae, the Cry1Ac toxin was largely resistant to digestion with protease K. Only about 60 amino acids were removed from the Cry1Ac amino terminus, which included primarily helix α1. Following incubation of the Cry1Ab or Cry1Ac toxins with vesicles, the preparations were solubilized by relatively mild conditions, and the toxin antigens were analyzed by immunoblotting. In both cases, most of the toxin formed a large, antigenic aggregate of ca. 200 kDa. These toxin aggregates did not include the toxin receptor aminopeptidase N, but interactions with other vesicle components were not excluded. No oligomerization occurred when inactive toxins with mutations in amphipathic helices (α5) and known to insert into the membrane were tested. Active toxins with other mutations in this helix did form oligomers. There was one exception; a very active helix α5 mutant toxin bound very well to membranes, but no oligomers were detected. Toxins with mutations in the loop connecting helices α2 and α3, which affected the irreversible binding to vesicles, also did not oligomerize. There was a greater extent of oligomerization of the Cry1Ac toxin with vesicles from the
Heliothis virescens
midgut than with those from the
M. sexta
midgut, which correlated with observed differences in toxicity. Tight binding of virtually the entire toxin molecule to the membrane and the subsequent oligomerization are both important steps in toxicity.
Microbial Control of the Culture ofArtemiaJuveniles through Preemptive Colonization by Selected Bacterial Strains
Authors: Verschuere Laurent , Rombaut Geert , Huys Geert , Dhont Jean , Sorgeloos Patrick , Verstraete Willy
Published: December 2019

The use of juvenileArtemiaas feed in aquaculture and in the pet shop industry has been getting more attention during the last decade. In this study, the use of selected bacterial strains to improve the nutritional value of dry food forArtemiajuveniles and to obtain control of the associated microbial community was examined. Nine bacterial strains were selected based on their positive effects on survival and/or growth ofArtemiajuveniles under monoxenic culture conditions, while other strains caused no significant effect, significantly lower rates of survival and/or growth, or even total mortality of theArtemia. The nine selected strains were used to preemptively colonize the culture water ofArtemiajuveniles. Xenic culture ofArtemiaunder suboptimal conditions yielded better survival and/or growth rates when they were grown in the preemptively colonized culture medium than when grown in autoclaved seawater. The preemptive colonization of the culture water had a drastic influence on the microbial communities that developed in the culture water or that were associated with theArtemia, as determined with Biolog GN community-level physiological profiles. Chemotaxonomical characterization based on fatty acid methyl ester analysis of bacterial isolates recovered from the culture tanks was performed, and a comparison with the initially introduced strains was made. Finally, several modes of action for the beneficial effect of the bacterial strains are proposed.
Chitinases from Uncultured Marine Microorganisms
Authors: Cottrell Matthew T. , Moore Jessica A. , Kirchman David L.
Published: December 2019

Our understanding of the degradation of organic matter will benefit from a greater appreciation for the genes encoding enzymes involved in the hydrolysis of biopolymers such as chitin, one of the most abundant polymers in nature. To isolate representative and abundant chitinase genes from uncultivated marine bacteria, we constructed libraries of genomic DNA isolated from coastal and estuarine waters. The libraries were screened for genes encoding proteins that hydrolyze a fluorogenic analogue of chitin, 4-methylumbelliferyl β-
d
-
N
,
N
′-diacetylchitobioside (MUF-diNAG). The abundance of clones capable of MUF-diNAG hydrolysis was higher in the library constructed with DNA from the estuary than in that constructed with DNA from coastal waters, although the abundance of positive clones was also dependent on the method used to screen the library. Plaque assays revealed nine MUF-diNAG-positive clones of 75,000 screened for the estuarine sample and two clones of 750,000 for the coastal sample. A microtiter plate assay revealed approximately 1 positive clone for every 500 clones screened in the coastal library. The number of clones detected with the plaque assay was consistent with estimates of the portion of culturable bacteria that degrade chitin. Our results suggest that culture-dependent methods do not greatly underestimate the portion of marine bacterial communities capable of chitin degradation.
Effect of Organic Solvents on the Yield of Solvent-Tolerant
Pseudomonas putida
S12
Authors: Isken Sonja , Derks Antoine , Wolffs Petra F. G. , de Bont Jan A. M.
Published: December 2019

Solvent-tolerant microorganisms are useful in biotransformations with whole cells in two-phase solvent-water systems. The results presented here describe the effects that organic solvents have on the growth of these organisms. The maximal growth rate of
Pseudomonas putida
S12, 0.8 h
−1
, was not affected by toluene in batch cultures, but in chemostat cultures the solvent decreased the maximal growth rate by nearly 50%. Toluene, ethylbenzene, propylbenzene, xylene, hexane, and cyclohexane reduced the biomass yield, and this effect depended on the concentration of the solvent in the bacterial membrane and not on its chemical structure. The dose response to solvents in terms of yield was linear up to an approximately 200 mM concentration of solvent in the bacterial membrane, both in the wild type and in a mutant lacking an active efflux system for toluene. Above this critical concentration the yield of the wild type remained constant at 0.2 g of protein/g of glucose with increasing concentrations of toluene. The reduction of the yield in the presence of solvents is due to a maintenance higher by a factor of three or four as well as to a decrease of the maximum growth yield by 33%. Therefore, energy-consuming adaptation processes as well as the uncoupling effect of the solvents reduce the yield of the tolerant cells.
cis/trans
Isomerase of Unsaturated Fatty Acids of
Pseudomonas putida
P8: Evidence for a Heme Protein of the Cytochrome
c
Type
Authors: Holtwick Rita , Keweloh Heribert , Meinhardt Friedhelm
Published: December 2019

From a pool of 600 temperature-sensitive transposon mutants of
Pseudomonas putida
P8, 1 strain was isolated that carries a mini-Tn
5
insertion within the cytochrome
c
operon. As a result, genes involved in the attachment of heme to cytochrome
c
-type proteins are turned off. Accordingly, cytochrome
c
could not be detected spectrophotometrically. The mutant also exhibited a remarkable reduction of
cis-trans
isomerization capability for unsaturated fatty acids. Consistent with the genetic and physiological data is the detection of a cytochrome
c
-type heme-binding motif close to the N terminus of the predicted polypeptide of the
cis/trans
isomerase (
cti
) gene (
C
VA
CH
; conserved amino acids in italics). The functional significance of this motif was proven by site-directed mutagenesis. A possible mechanism of heme-catalyzed
cis-trans
isomerization of unsaturated fatty acids is discussed.
PCR-Based Genotyping of Epidemic and Preepidemic
Trichoderma
Isolates Associated with Green Mold of
Agaricus bisporus
Authors: Chen X. , Romaine C. P. , Tan Q. , Schlagnhaufer B. , Ospina-Giraldo M. D. , Royse D. J. , Huff D. R.
Published: December 2019

We used randomly amplified polymorphic DNA (RAPD)-PCR to estimate genetic variation among isolates of
Trichoderma
associated with green mold on the cultivated mushroom
Agaricus bisporus
. Of 83 isolates examined, 66 were sampled during the recent green mold epidemic, while the remaining 17 isolates were collected just prior to the epidemic and date back to the 1950s.
Trichoderma harzianum
biotype 4 was identified by RAPD analysis as the cause of almost 90% of the epidemic-related episodes of green mold occurring in the major commercial mushroom-growing region in North America. Biotype 4 was more closely allied to
T. harzianum
biotype 2, the predominant pathogenic genotype in Europe, than to the less pathogenic biotype 1 and
Trichoderma atroviride
(formerly
T. harzianum
biotype 3). No variation in the RAPD patterns was observed among the isolates within biotype 2 or 4, suggesting that the two pathogenic biotypes were populations containing single clones. Considerable genetic variation, however, was noted among isolates of biotype 1 and
T. atroviride
from Europe. Biotype 4 was not represented by the preepidemic isolates of
Trichoderma
as determined by RAPD markers and PCR amplification of an arbitrary DNA sequence unique to the genomes of biotypes 2 and 4. Our findings suggest that the onset of the green mold epidemic in North America resulted from the recent introduction of a highly virulent genotype of the pathogen into cultivated mushrooms.
Increase in Alfalfa Nodulation, Nitrogen Fixation, and Plant Growth by Specific DNA Amplification in
Sinorhizobium meliloti
Authors: Castillo Marcela , Flores Margarita , Mavingui Patrick , Martínez-Romero Esperanza , Palacios Rafael , Hernández Georgina
Published: December 2019

To improve symbiotic nitrogen fixation on alfalfa plants,
Sinorhizobium meliloti
strains containing different average copy numbers of a symbiotic DNA region were constructed by specific DNA amplification (SDA). A DNA fragment containing a regulatory gene (
nodD1
), the common nodulation genes (
nodABC
), and an operon essential for nitrogen fixation (
nifN
) from the
nod
regulon region of the symbiotic plasmid pSyma of
S. meliloti
was cloned into a plasmid unable to replicate in this organism. The plasmid then was integrated into the homologous DNA region of
S. meliloti
strains 41 and 1021, which resulted in a duplication of the symbiotic region.
Sinorhizobium
derivatives carrying further amplification were selected by growing the bacteria in increased concentrations of an antibiotic marker present in the integrated vector. Derivatives of strain 41 containing averages of 3 and 6 copies and a derivative of strain 1021 containing an average of 2.5 copies of the symbiotic region were obtained. In addition, the same region was introduced into both strains as a multicopy plasmid, yielding derivatives with an average of seven copies per cell. Nodulation, nitrogenase activity, plant nitrogen content, and plant growth were analyzed in alfalfa plants inoculated with the different strains. The copy number of the symbiotic region was critical in determining the plant phenotype. In the case of the strains with a moderate increase in copy number, symbiotic properties were improved significantly. The inoculation of alfalfa with these strains resulted in an enhancement of plant growth.
Iron-Oxidizing Bacteria Are Associated with Ferric Hydroxide Precipitates (Fe-Plaque) on the Roots of Wetland Plants
Authors: Emerson David , Weiss Johanna V. , Megonigal J. Patrick
Published: December 2019

The presence of Fe-oxidizing bacteria in the rhizosphere of four different species of wetland plants was investigated in a diverse wetland environment that had Fe(II) concentrations ranging from tens to hundreds of micromoles per liter and a pH range of 3.5 to 6.8. Enrichments for neutrophilic, putatively lithotrophic Fe-oxidizing bacteria were successful on roots from all four species; acidophilic Fe-oxidizing bacteria were enriched only on roots from plants whose root systems were exposed to soil solutions with a pH of <4. In
Sagittaria australis
there was a positive correlation (
P
< 0.01) between cell numbers and the total amount of Fe present; the same correlation was not found for
Leersia oryzoides
. These results present the first evidence for culturable Fe-oxidizing bacteria associated with Fe-plaque in the rhizosphere.
Virus Passage through Track-Etch Membranes Modified by Salinity and a Nonionic Surfactant
Authors: Lytle C. David , Routson Licia B. , Jain Nisha B. , Myers Matthew R. , Green Barbara L.
Published: December 2019

Why do viruses sometimes not pass through larger pores in track-etch filters? Increasing the salinity (0.8 to 160 mM Na
+
) decreased φX174 and PRD1 passage through track-etch polycarbonate membranes (sodium dodecyl sulfate coated but not polyvinylpyrrolidone coated) and PRD1 passage through polyester membranes. Undiminished passage when 0.1% Tween 80 was added implied that nonionic virus adsorption occurred and indicated that high levels of salinity decreased virus passage by decreasing electrostatic repulsion that prevented adsorption.
Mechanism of Alanine Hyperproduction by
Arthrobacter oxydans
HAP-1: Metabolic Shift to Fermentation under Nongrowth Aerobic Conditions
Authors: Hashimoto Shin-ichi , Katsumata Ryoichi
Published: December 2019

Arthrobacter oxydans
HAP-1 hyperproduces
dl
-alanine in a non-growth-associated manner. We found that decreased activities of pyruvate dehydrogenase and of the enzyme catalyzing NADH oxidation in the stationary phase are paralleled by a shift of pyruvate metabolism to alanine synthesis by
l
-alanine dehydrogenase. We propose that this enzyme functions as an electron sink even under aerobic conditions.
High-Efficiency Transformation of
Rhizobium leguminosarum
by Electroporation
Authors: Garg Bindu , Dogra Romesh C. , Sharma Parveen K.
Published: December 2019

Electrotransformation of
Rhizobium leguminosarum
was successfully carried out with a 15.1-kb plasmid, pMP154 (Cm
r
), containing a
nodABC-lacZ
fusion by electroporation. The maximum transformation efficiency, 10
8
transformants/μg of DNA, was achieved at a field strength of 14 kV/cm with a pulse of 7.3 ms (186 Ω). The number of transformants was found to increase with increasing cell density, with no sign of saturation. In relation to DNA dosage, the maximum transformation efficiency (5.8 × 10
8
transformants/μg of DNA) was obtained with 0.5 μg of DNA/ml of cell suspension, and a further increase in the DNA concentration resulted in a decline in transformation efficiency.