bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Holistic Approach for Determining the Entomopathogenic Potential of
Bacillus thuringiensis
Strains
Authors: Masson Luke , Erlandson Martin , Puzstai-Carey Marianne , Brousseau Roland , Juárez-Pérez Victor , Frutos Roger 
Published: December 2019

ABSTRACT

The
cry
gene content of
Bacillus thuringiensis
subsp.
aizawai
HD-133 was analyzed by a combination of high-pressure liquid chromatography (HPLC) and exclusive PCR. A total of six
cry
genes were detected in genomic DNA purified from HD-133, four from the
cry1
family (
cry1Aa
,
cry1Ab
,
cry1C
, and
cry1D
) as well as a gene each from the
cry2
(
cry2B
) and the
cry1I
families. To directly determine which genes were expressed and crystallized in the purified parasporal inclusions, solubilized and trypsinized HD-133 crystals were subjected to chromatographic separation by HPLC. Only three proteins, Cry1Ab, Cry1C, and Cry1D, were found, in a 60/37/3 ratio. Dot blot analysis of total mRNA purified from HD-133 showed that both the
cry2B
and
cry1I
genes, but not the
cry1Aa
gene, were transcribed. Cloning and sequencing of the
cry1Aa
gene revealed an inserted DNA sequence within the
cry
coding sequence, resulting in a disrupted reading frame. Taken together, our results show that combining crystal protein analysis with a genetic approach is a highly complementary and powerful way to assess the potential of
B. thuringiensis
isolates for new insecticidal genes and specificities. Furthermore, based on the number of cryptic genes found in HD-133, the total
cry
gene content of
B. thuringiensis
strains may be higher than previously thought.
Procaryotic Expression of Single-Chain Variable-Fragment (scFv) Antibodies: Secretion in L-Form Cells of
Proteus mirabilis
Leads to Active Product and Overcomes the Limitations of Periplasmic Expression in
Escherichia coli
Authors: Rippmann Jörg F. , Klein Michaela , Hoischen Christian , Brocks Bodo , Rettig Wolfgang J. , Gumpert Johannes , Pfizenmaier Klaus , Mattes Ralf , Moosmayer Dieter 
Published: December 2019

ABSTRACT

Recently it has been demonstrated that L-form cells of
Proteus mirabilis
(L VI), which lack a periplasmic compartment, can be efficiently used in the production and secretion of heterologous proteins. In search of novel expression systems for recombinant antibodies, we compared levels of single-chain variable-fragment (scFv) production in
Escherichia coli
JM109 and
P. mirabilis
L VI, which express four distinct scFvs of potential clinical interest that show differences in levels of expression and in their tendencies to form aggregates upon periplasmic expression. Production of all analyzed scFvs in
E. coli
was limited by the severe toxic effect of the heterologous product as indicated by inhibition of culture growth and the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. In contrast, the L-form cells exhibited nearly unlimited growth under the tested production conditions for all scFvs examined. Moreover, expression experiments with
P. mirabilis
L VI led to scFv concentrations in the range of 40 to 200 mg per liter of culture medium (corresponding to volume yields 33- to 160-fold higher than those with
E. coli
JM109), depending on the expressed antibody. In a translocation inhibition experiment the secretion of the scFv constructs was shown to be an active transport coupled to the signal cleavage. We suppose that this direct release of the newly synthesized product into a large volume of the growth medium favors folding into the native active structure. The limited aggregation of scFv observed in the
P. mirabilis
L VI supernatant (occurring in a first-order-kinetics manner) was found to be due to intrinsic features of the scFv and not related to the expression process of the host cells. The
P. mirabilis
L VI supernatant was found to be advantageous for scFv purification. A two-step chromatography procedure led to homogeneous scFv with high antigen binding activity as revealed from binding experiments with eukaryotic cells.
Characterization of Cell Lysis in
Pseudomonas putida
Induced upon Expression of Heterologous Killing Genes
Authors: Ronchel M. Carmen , Molina Lázaro , Witte Angela , Lutbiz Werner , Molin Søren , Ramos Juan L. , Ramos Cayo 
Published: December 2019

ABSTRACT

Active biological containment systems are based on the controlled expression of killing genes. These systems are of interest for the
Pseudomonadaceae
because of the potential applications of these microbes as bioremediation agents and biopesticides. The physiological effects that lead to cell death upon the induction of expression of two different heterologous killing genes in nonpathogenic
Pseudomonas putida
KT2440 derivatives have been analyzed.
P. putida
CMC4 and CMC12 carry in their chromosomes a fusion of the P
A1-04/03
promoter to the
Escherichia coli gef
gene and the φX174 lysis gene
E
, respectively. Expression of the killing genes is controlled by the LacI protein, whose expression is initiated from the XylS-dependent Pm promoter. Under induced conditions, killing of
P. putida
CMC12 cells mediated by φX174 lysis protein E was faster than that observed for
P. putida
CMC4, for which the Gef protein was the killing agent. In both cases, cell death occurred as a result of impaired respiration, altered membrane permeability, and the release of some cytoplasmic contents to the extracellular medium.
Detection and Isolation of Novel Rhizopine-Catabolizing Bacteria from the Environment
Authors: Gardener Brian B. McSpadden , de Bruijn Frans J. 
Published: December 2019

ABSTRACT

Microbial rhizopine-catabolizing (Moc) activity was detected in serial dilutions of soil and rhizosphere washes. The activity observed generally ranged between 10
6
and 10
7
catabolic units per g, and the numbers of nonspecific culture-forming units were found to be approximately 10 times higher. A diverse set of 37 isolates was obtained by enrichment on
scyllo
-inosamine-containing media. However, none of the bacteria that were isolated were found to contain DNA sequences homologous to the known
mocA
,
mocB
, and
mocC
genes of
Sinorhizobium meliloti
L5-30. Twenty-one of the isolates could utilize an SI preparation as the sole carbon and nitrogen source for growth. Partial sequencing of 16S ribosomal DNAs (rDNAs) amplified from these strains indicated that five distinct bacterial genera (
Arthrobacter
,
Sinorhizobium
,
Pseudomonas
,
Aeromonas
, and
Alcaligenes
) were represented in this set. Only 6 of these 21 isolates could catabolize 3-
O
-methyl-
scyllo
-inosamine under standard assay conditions. Two of these, strains D1 and R3, were found to have 16S rDNA sequences very similar to those of
Sinorhizobium meliloti
. However, these strains are not symbiotically effective on
Medicago sativa
, and DNA sequences homologous to the
nodB
and
nodC
genes were not detected in strains D1 and R3 by Southern hybridization analysis.
Flow Cytometric Analysis of the In Situ Accessibility of
Escherichia coli
16S rRNA for Fluorescently Labeled Oligonucleotide Probes
Authors: Fuchs Bernhard Maximilian , Wallner Günter , Beisker Wolfgang , Schwippl Ines , Ludwig Wolfgang , Amann Rudolf 
Published: December 2019

ABSTRACT

In situ identification of whole fixed bacterial cells by hybridization with fluorescently labeled, rRNA-targeted oligonucleotide probes is often limited by low signal intensities. In addition to an impermeability of the cell periphery and a low cellular rRNA content, the three-dimensional structure of the ribosome may hinder the access of oligonucleotides to their target sites. Until now, a systematic study on the accessibility of 16S rRNA target sites had not been done. Here, we report fluorescence intensities obtained with more than 200 oligonucleotide probes (mostly 18-mers) used with whole fixed cells of
Escherichia coli
DSM 30083
T
. Two overlapping sets of adjacent oligonucleotides, 171 in total, were designed to cover the full length of the 16S rRNA. The two sets are shifted by 5 to 13 nucleotides. The probes were labeled with carboxyfluorescein, and signal intensities of hybridized cells were quantified by flow cytometry. Care was taken that the signal intensity of cells was dependent solely on the in situ accessibility of probe target sites. The brightest signal resulted from probe Eco1482, complementary to positions 1482 to 1499. With this probe, the fluorescence was 1.7 times brighter than that of the standard bacterial probe EUB338 and 44 times brighter than that of the worst probe, Eco468. The distribution of probe-conferred cell fluorescence in six arbitrarily set brightness classes (classes I to VI; 100 to 81%, 80 to 61%, 60 to 41%, 40 to 21%, 20 to 6%, and 5 to 0% of the brightness with Eco1482, respectively) was as follows: I, 4%; II, 14%; III, 21%; IV, 29%, V, 19%; and VI, 13%. A more detailed analysis of helices 6, 18, and 23 with additional probes demonstrated that a shift of the target region by only a few bases could result in a decline of cell fluorescence from >80 to <10%. Considering the high evolutionary conservation of 16S rRNA, the in situ accessibility map of
E. coli
should facilitate a more rational selection of probe target sites for other species as well.
Phylogenetic Affiliation of Soil Bacteria That Degrade Aliphatic Polyesters Available Commercially as Biodegradable Plastics
Authors: Suyama Tetsushi , Tokiwa Yutaka , Ouichanpagdee Pornpimol , Kanagawa Takahiro , Kamagata Yoichi 
Published: December 2019

ABSTRACT

Thirty-nine morphologically different soil bacteria capable of degrading poly(β-hydroxyalkanoate), poly(ɛ-caprolactone), poly(hexamethylene carbonate), or poly(tetramethylene succinate) were isolated. Their phylogenetic positions were determined by 16S ribosomal DNA sequencing, and all of them fell into the classes
Firmicutes
and
Proteobacteria
. Determinations of substrate utilization revealed characteristic patterns of substrate specificities.
Effects of Bacterial Antibiotic Production on Rhizosphere Microbial Communities from a Culture-Independent Perspective
Authors: Robleto Eduardo A. , Borneman James , Triplett Eric W. 
Published: December 2019

ABSTRACT

The effects of antibiotic production on rhizosphere microbial communities of field-grown
Phaseolus vulgaris
were assessed by using ribosomal intergenic spacer analysis. Inoculum strains of
Rhizobium etli
CE3 differing only in trifolitoxin production were used. Trifolitoxin production dramatically reduced the diversity of trifolitoxin-sensitive members of the α subdivision of the class
Proteobacteria
with little apparent effect on most microbes.
Effects of Halides on Plasmid-Mediated Silver Resistance in
Escherichia coli
Authors: Gupta Amit , Maynes Maria , Silver Simon 
Published: December 2019

ABSTRACT

Silver resistance of sensitive
Escherichia coli
J53 and resistance plasmid-containing J53(pMG101) was affected by halides in the growth medium. The effects of halides on Ag
+
resistance were measured with AgNO
3
and silver sulfadiazine, both on agar and in liquid. Low concentrations of chloride made the differences in MICs between sensitive and resistant strains larger. High concentrations of halides increased the sensitivities of both strains to Ag
+
.
Influence of Temperature and Pressure on the Lethality of Ultrasound
Authors: Raso J. , Pagán R. , Condón S. , Sala F. J. 
Published: December 2019

ABSTRACT

A specially designed resistometer was constructed, and the lethal effect on
Yersinia enterocolitica
of ultrasonic waves (UW) at different static pressures (manosonication [MS]) and of combined heat-UW under pressure treatments (manothermosonication [MTS]) was investigated. During MS treatments at 30°C and 200 kPa, the increase in the amplitude of UW of 20 kHz from 21 to 150 μm exponentially decreased decimal reduction time values (
D
MS
) from 4 to 0.37 min. When pressure was increased from 0 to 600 kPa at a constant amplitude (150 μm) and temperature (30°C),
D
MS
values decreased from 1.52 to 0.20 min. The magnitude of this decrease in
D
MS
declined progressively as pressure was increased. The influence of pressure on
D
MS
values was greater with increased amplitude of UW. Pressure alone of as much as 600 kPa did not influence the heat resistance of
Y. enterocolitica
(
D
60
= 0.094;
z
= 5.65). At temperatures of as much as 58°C, the lethality of UW under pressure was greater than that of heat treatment alone at the same temperature. At higher temperatures, this difference disappeared. Heat and UW under pressure seemed to act independently. The lethality of MTS treatments appeared to result from the added effects of UW under pressure and the lethal effect of heat. The individual contributions of heat and of UW under pressure to the total lethal effect of MTS depended on temperature. The inactivating effect of UW was not due to titanium particles eroded from the sonication horn. The addition to the MS media of cysteamine did not increase the resistance of
Y. enterocolitica
to MS treatment. MS treatment caused cell disruption.
A Cold-Adapted Lipase of an Alaskan Psychrotroph,
Pseudomonas
sp. Strain B11-1: Gene Cloning and Enzyme Purification and Characterization
Authors: Choo Dong-Won , Kurihara Tatsuo , Suzuki Takeshi , Soda Kenji , Esaki Nobuyoshi 
Published: December 2019

ABSTRACT

A psychrotrophic bacterium producing a cold-adapted lipase upon growth at low temperatures was isolated from Alaskan soil and identified as a
Pseudomonas
strain. The lipase gene (
lipP
) was cloned from the strain and sequenced. The amino acid sequence deduced from the nucleotide sequence of the gene (924 bp) corresponded to a protein of 308 amino acid residues with a molecular weight of 33,714. LipP also has consensus motifs conserved in other cold-adapted lipases, i.e., Lipase 2 from Antarctic
Moraxella
TA144 (G. Feller, M. Thiry, J. L. Arpigny, and C. Gerday, DNA Cell Biol. 10:381–388, 1991) and the mammalian hormone-sensitive lipase (D. Langin, H. Laurell, L. S. Holst, P. Belfrage, and C. Holm, Proc. Natl. Acad. Sci. USA 90:4897–4901, 1993): a pentapeptide, GDSAG, containing the putative active-site serine and an HG dipeptide. LipP was purified from an extract of recombinant
Escherichia coli
C600 cells harboring a plasmid coding for the
lipP
gene. The enzyme showed a 1,3-positional specificity toward triolein.
p
-Nitrophenyl esters of fatty acids with short to medium chains (C
4
and C
6
) served as good substrates. The enzyme was stable between pH 6 and 9, and the optimal pH for the enzymatic hydrolysis of tributyrin was around 8. The activation energies for the hydrolysis of
p
-nitrophenyl butyrate and
p
-nitrophenyl laurate were determined to be 11.2 and 7.7 kcal/mol, respectively, in the temperature range 5 to 35°C. The enzyme was unstable at temperatures higher than 45°C. The

K
m

of the enzyme for
p
-nitrophenyl butyrate increased with increases in the assay temperature. The enzyme was strongly inhibited by Zn
2+
, Cu
2+
, Fe
3+
, and Hg
2+
but was not affected by phenylmethylsulfonyl fluoride and bis-nitrophenyl phosphate. Various water-miscible organic solvents, such as methanol and dimethyl sulfoxide, at concentrations of 0 to 30% (vol/vol) activated the enzyme.
Engineering of a Cold-Adapted Protease by Sequential Random Mutagenesis and a Screening System
Authors: Taguchi Seiichi , Ozaki Akiyoshi , Momose Haruo 
Published: December 2019

ABSTRACT

A cold-adapted protease subtilisin was successfully isolated by evolutionary engineering based on sequential in vitro random mutagenesis and an improved method of screening (H. Kano, S. Taguchi, and H. Momose, Appl. Microbiol. Biotechnol. 47:46–51, 1997). The mutant subtilisin, termed m-63, exhibited a catalytic efficiency (expressed as the
k
cat
/

K
m

value) 100% higher than that of the wild type at 10°C when
N
-succinyl-
l
-Ala-
l
-Ala-
l
-Pro-
l
-Phe-
p
-nitroanilide was used as a synthetic substrate. This cold adaptation was achieved with three mutations, Val to Ile at position 72 (V72I), Ala to Thr at position 92 (A92T), and Gly to Asp at position 131 (G131D), and it was found that an increase in substrate affinity (i.e., a decreased

K
m

value) was mostly responsible for the increased activity. Analysis of kinetic parameters revealed that the V72I mutation contributed negatively to the activity but that the other two mutations, A92T and G131D, overcame the negative contribution to confer the 100% increase in activity. Besides suppression of the activity-negative mutation (V72I) by A92T and G131D, suppression of structural stability was observed in measurements of activity retention at 60°C and circular dichroism spectra at 10°C.
Immunomagnetic Capture PCR for Rapid Concentration and Detection of Hepatitis A Virus from Environmental Samples
Authors: Jothikumar N. , Cliver Dean O. , Mariam Tadesse W. 
Published: December 2019

ABSTRACT
We studied the concentration of hepatitis A virus (HAV) from environmental samples by membrane filter-based urea-arginine phosphate buffer and its detection by using immunomagnetic capture (IC) reverse transcription (RT)-PCR (IC PCR). Magnetic beads coated with anti-HAV rabbit antibodies were used for enrichment and concentration of HAV from environmental samples. IC PCR is sensitive enough to detect as few as 0.04 PFU of cell culture-adapted HAV in inoculated water and sewage samples. IC PCR is specific and does not yield positive reactions with poliovirus 1, HAV RNA, or selected bacteriophages. IC concentrates viruses suspended in small volumes to microliter volumes that can be used directly in RT-PCR. IC concentration of viruses from sewage samples without concentration of inhibitory substances is important for successful RT-PCR detection. In a field trial, 2 of 18 raw sewage samples tested by IC PCR were positive for HAV.
The Use of Fluorogenic Substrates To Measure Fungal Presence and Activity in Soil
Authors: Miller Morten , Palojärvi Ansa , Rangger Andrea , Reeslev Morten , Kjøller Annelise 
Published: December 2019

ABSTRACT

Our objective was to determine if 4-methylumbelliferyl-labelled enzyme substrates could be used to detect and quantify specific components of chitinase and cellulase activities as specific indicators of the presence and activity of fungal biomass. The fluorogenic substrates 4-methylumbelliferyl (MUF)
N
-acetyl-β-
d
-glucosaminide and MUF β-
d
-lactoside were used for the detection and quantification of β-
N
-acetylglucosaminidase (EC
3.2.1.30
) (NAGase) and endo 1,4-β-glucanase (EC
3.2.1.4
)/cellobiohydrolase (EC
3.2.1.91
) (CELase), respectively. Culture screenings on solid media showed a widespread ability to produce NAGase among a taxonomically diverse selection of fungi on media with and without added chitin. NAGase activity was expressed only in a limited number of bacteria and on media supplemented with chitin. The CELase activity was observed only in a limited number of fungi and bacteria. Bacterial CELase activity was expressed on agar media containing a cellulose-derived substrate. In soil samples, NAGase activity was significantly correlated with estimates of fungal biomass, based on the content of two fungus-specific indicator molecules, 18:2ω6 phospholipid fatty acid (PLFA) and ergosterol. CELase activity was significantly correlated with the PLFA-based estimate of fungal biomass in the soil, but no correlation was found with ergosterol-based estimates of fungal biomass.
Size-Selective Predation on Groundwater Bacteria by Nanoflagellates in an Organic-Contaminated Aquifer
Authors: Kinner N. E. , Harvey R. W. , Blakeslee K. , Novarino G. , Meeker L. D. 
Published: December 2019

ABSTRACT

Time series incubations were conducted to provide estimates for the size selectivities and rates of protistan grazing that may be occurring in a sandy, contaminated aquifer. The experiments involved four size classes of fluorescently labeled groundwater bacteria (FLB) and 2- to 3-μm-long nanoflagellates, primarily
Spumella guttula
(Ehrenberg) Kent, that were isolated from contaminated aquifer sediments (Cape Cod, Mass.). The greatest uptake and clearance rates (0.77 bacteria · flagellate
−1
· h
−1
and 1.4 nl · flagellate
−1
· h
−1
, respectively) were observed for 0.8- to 1.5-μm-long FLB (0.21-μm
3
average cell volume), which represent the fastest growing bacteria within the pore fluids of the contaminated aquifer sediments. The 19:1 to 67:1 volume ratios of nanoflagellate predators to preferred bacterial prey were in the lower end of the range commonly reported for other aquatic habitats. The grazing data suggest that the aquifer nanoflagellates can consume as much as 12 to 74% of the unattached bacterial community in 1 day and are likely to have a substantive effect upon bacterial degradation of organic groundwater contaminants.
Reverse Transcriptase (RT) Inhibition of PCR at Low Concentrations of Template and Its Implications for Quantitative RT-PCR
Authors: Chandler Darrell P. , Wagnon Christina A. , Bolton Harvey 
Published: December 2019

ABSTRACT

Numerous instances of reverse transcriptase (RT) inhibition of the PCR were observed while developing nonquantitative uncoupled RT-PCR techniques for detecting nitrogenase and ammonia monooxygenase gene expression in situ. The inhibitory effect of RT on the PCR was removed with increasing template concentrations beyond 10
5
to 10
6
copies. Including T4 gene 32 protein during the reverse transcription phase of the RT-PCR reaction increased the RT-PCR product yield by as much as 483%; if gene 32 protein was introduced after reverse transcription but prior to the PCR phase, no improvement in product yield was observed. Addition of 1 μg of exogenous calf thymus DNA or yeast tRNA did little to relieve RT inhibition of the PCR on both genomic DNA and mRNA templates. These results suggest that RT inhibition of the PCR is mediated through direct interaction with the specific primer-template combination (DNA and RNA) and point to specific assay modifications for estimating the extent of RT inhibition and counteracting some of the inhibitory effect. Furthermore, the working hypothesis of RT inhibition below a 10
5
to 10
6
copy threshold has important implications for quantitative RT-PCR studies. In particular, competitive, quantitative RT-PCR systems will consistently underestimate the actual RNA concentration. Hence, enumerations of RNA templates below 10
5
to 10
6
copies will be relative to an internal standard and will not be an absolute measure of RNA abundance in situ.
In Situ Gene Expression in Mixed-Culture Biofilms: Evidence of Metabolic Interactions between Community Members
Authors: Møller Søren , Sternberg Claus , Andersen Jens Bo , Christensen Bjarke Bak , Ramos Juan Luis , Givskov Michael , Molin Søren 
Published: December 2019

ABSTRACT

Microbial communities growing in laboratory-based flow chambers were investigated in order to study compartmentalization of specific gene expression. Among the community members studied, the focus was in particular on
Pseudomonas putida
and a strain of an
Acinetobacter
sp., and the genes studied are involved in the biodegradation of toluene and related aromatic compounds. The upper-pathway promoter (
Pu
) and the
meta
-pathway promoter (
Pm
) from the TOL plasmid were fused independently to the gene coding for the green fluorescent protein (GFP), and expression from these promoters was studied in
P. putida
, which was a dominant community member. Biofilms were cultured in flow chambers, which in combination with scanning confocal laser microscopy allowed direct monitoring of promoter activity with single-cell spatial resolution. Expression from the
Pu
promoter was homogeneously induced by benzyl alcohol in both community and pure-culture biofilms, while the
Pm
promoter was induced in the mixed community but not in a pure-culture biofilm. By sequentially adding community members, induction of
Pm
was shown to be a consequence of direct metabolic interactions between an
Acinetobacter
species and
P. putida
. Furthermore, in fixed biofilm samples organism identity was determined and gene expression was visualized at the same time by combining GFP expression with in situ hybridization with fluorescence-labeled 16S rRNA targeting probes. This combination of techniques is a powerful approach for investigating structure-function relationships in microbial communities.