bluuebunny/crossref_metadata_2025_split huggingface dataset:
Engineering of a Single-Chain Variable-Fragment (scFv) Antibody Specific for the Stolbur Phytoplasma (Mollicute) and Its Expression in
Escherichia coli
and Tobacco Plants
Authors: Le Gall Fabrice , Bové Joseph-Marie , Garnier Monique 
Published: December 2019

ABSTRACT

From a hybridoma cell line (2A10) producing an immunoglobulin G1 directed against the major membrane protein of the stolbur phytoplasma, we have engineered scFv (single-chain variable-fragment) antibodies from the variable heavy (VH) and light (VL) domains of the immunoglobulin. The scFv gene was cloned and expressed in
Escherichia coli
. The expressed protein of 30 kDa could be recovered from the periplasmic fraction of the bacterial cells and was shown to be fully functional toward its phytoplasmal antigen, since enzyme-linked immunosorbent assay or immunofluorescence (IF) detection of the stolbur phytoplasma antigen by the scFv was identical to that of the native immunoglobulin. The scFv gene was then cloned in plasmid pBG-dAb-BIN of
Agrobacterium tumefaciens
to transform tobacco plants. The transformed plants were screened by PCR and Northern blotting for the presence and expression of the transgene, respectively, and by IF for expression of the scFv. One transgenic tobacco line, 1A6, was selected for challenge inoculation with the stolbur phytoplasma. When grafted on a stolbur phytoplasma-infected tobacco rootstock, the transgenic tobacco shoots grew free of symptoms and flowered after 2 months, while normal tobacco shoots showed severe stolbur symptoms during the same period and eventually died.
Design of a Phage-Insensitive Lactococcal Dairy Starter via Sequential Transfer of Naturally Occurring Conjugative Plasmids
Authors: O’Sullivan David , Coffey Aidan , Fitzgerald Gerald F. , Hill Colin , Ross R. Paul 
Published: December 2019

ABSTRACT

The plasmid-free
Lactococcus lactis
subsp.
cremoris
MG1614 is highly phage sensitive and lacks lactose fermenting ability (Lac) and primary casein degrading ability (Prt). Food grade gene transfer systems were used to sequentially superimpose different phage defense systems on this background, resulting in a gradual increase in resistance to bacteriophage in the derivatives. pLP712, encoding Lac and Prt, was then transferred to one of these hosts, into which plasmids encoding adsorption inhibition, restriction modification, and abortive infection had already been introduced. This resulted in a phage-resistant strain which was successfully used as a single-strain starter for cheddar cheese manufacture under industrial conditions.
A Polyphasic Approach To Study the Diversity and Vertical Distribution of Sulfur-Oxidizing
Thiomicrospira
Species in Coastal Sediments of the German Wadden Sea
Authors: Brinkhoff Thorsten , Santegoeds Cecilia M. , Sahm Kerstin , Kuever Jan , Muyzer Gerard 
Published: December 2019

ABSTRACT

Recently, four
Thiomicrospira
strains were isolated from a coastal mud flat of the German Wadden Sea (T. Brinkhoff and G. Muyzer, Appl. Environ. Microbiol. 63:3789–3796, 1997). Here we describe the use of a polyphasic approach to investigate the functional role of these closely related bacteria. Microsensor measurements showed that there was oxygen penetration into the sediment to a depth of about 2.0 mm. The pH decreased from 8.15 in the overlaying water to a minimum value of 7.3 at a depth of 1.2 mm. Further down in the sediment the pH increased to about 7.8 and remained constant. Most-probable-number (MPN) counts of chemolithoautotrophic sulfur-oxidizing bacteria revealed nearly constant numbers along the vertical profile; the cell concentration ranged from 0.93 × 10
5
to 9.3 × 10
5
cells per g of sediment. A specific PCR was used to detect the presence of
Thiomicrospira
cells in the MPN count preparations and to determine their 16S rRNA sequences. The concentration of
Thiomicrospira
cells did not decrease with depth. It was found that
Thiomicrospira
strains were not dominant sulfur-oxidizing bacteria in this habitat. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA fragments followed by hybridization analysis with a genus-specific oligonucleotide probe revealed the diversity of
Thiomicrospira
strains in the MPN cultures. Sequence analysis of the highest MPN dilutions in which the genus
Thiomicrospira
was detected revealed that there were four clusters of several closely related sequences. Only one of the 10
Thiomicrospira
sequences retrieved was related to sequences of known isolates from the same habitat. Slot blot hybridization of rRNA isolated from different sediment layers showed that, in contrast to the concentration of
Thiomicrospira
cells, the concentration of
Thiomicrospira
-specific rRNA decreased rapidly in the region below the oxic layer of the sediment. This study revealed the enormous sequence diversity of closely related microorganisms present in one habitat, which so far has been found only by sequencing molecular isolates. In addition, it showed that most of the
Thiomicrospira
populations in the sediment studied were quiescent.
In Vivo Transposon Mutagenesis in
Haemophilus influenzae
Authors: Kraiß Anita , Schlör Stefan , Reidl Joachim 
Published: December 2019

ABSTRACT

In order to devise an in vivo insertion mutagenesis scheme for
Haemophilus influenzae
, a novel set of transposons has been constructed. These are Tn
10
-based minitransposons carried on pACYC184- and pACYC177-based replicons, which are suitable for in vivo transposition in
H. influenzae
. The transposon delivery system was designed to contain an
H. influenzae
-specific uptake signal sequence which facilitates DNA transformation into
H. influenzae
. The following mini-Tn
10
elements have been made suitable for specific tasks in
H. influenzae
: (i) Tn
10d-cat
, which can be used to generate chloramphenicol-selectable insertion mutations; (ii) Tn
10d-bla
, an ampicillin-selectable translational fusion system allowing the detection of membrane or secreted proteins; and (iii) Tn
10d-lacZcat
, a chloramphenicol-selectable
lacZ
transcriptional fusion system. For the rapid identification of the transposon insertions, a PCR fragment enrichment method was developed. This report demonstrates that this in vivo mutagenesis technique is a convenient tool for the analysis of biochemical and regulatory pathways in the human pathogen
H. influenzae
.
Molecular Cloning and Functional Expression in
Lactobacillus plantarum
80 of
xylT
, Encoding the
d
-Xylose–H
+
Symporter of
Lactobacillus brevis
Authors: Chaillou Stéphane , Bor Yeou-Cherng , Batt Carl A. , Postma Pieter W. , Pouwels Peter H. 
Published: December 2019

ABSTRACT

A 3-kb region, located downstream of the
Lactobacillus brevis xylA
gene (encoding
d
-xylose isomerase), was cloned in
Escherichia coli
TG1. The sequence revealed two open reading frames which could code for the
d
-xylulose kinase gene (
xylB
) and another gene (
xylT
) encoding a protein of 457 amino acids with significant similarity to the
d
-xylose–H
+
symporters of
E. coli
, XylE (57%), and
Bacillus megaterium
, XylT (58%), to the
d
-xylose–Na
+
symporter of
Tetragenococcus halophila
, XylE (57%), and to the
l
-arabinose–H
+
symporter of
E. coli
, AraE (60%). The
L. brevis xylABT
genes showed an arrangement similar to that of the
B. megaterium xylABT
operon and the
T. halophila xylABE
operon. Southern hybridization performed with the
Lactobacillus pentosus xylR
gene (encoding the
d
-xylose repressor protein) as a probe revealed the existence of a
xylR
homologue in
L. brevis
which is not located with the
xyABT
locus. The existence of a functional XylR was further suggested by the presence of
xylO
sequences upstream of
xylA
and
xylT
and by the requirement of
d
-xylose for the induction of
d
-xylose isomerase,
d
-xylulose kinase, and
d
-xylose transport activities in
L. brevis
. When
L. brevis
was cultivated in a mixture of
d
-glucose and
d
-xylose, the
d
-xylose isomerase and
d
-xylulose kinase activities were reduced fourfold and the
d
-xylose transport activity was reduced by sixfold, suggesting catabolite repression by
d
-glucose of
d
-xylose assimilation. The
xylT
gene was functionally expressed in
Lactobacillus plantarum
80, a strain which lacks proton motive force-linked
d
-xylose transport activity. The role of the XylT protein was confirmed by the accumulation of
d
-xylose in
L. plantarum
80 cells, and this accumulation was dependent on the proton motive force generated by either malolactic fermentation or by the metabolism of
d
-glucose. The apparent affinity constant of XylT for
d
-xylose was approximately 215 μM, and the maximal initial velocity of transport was 35 nmol/min per mg (dry weight). Furthermore, of a number of sugars tested, only 6-deoxy-
d
-glucose inhibited the transport of
d
-xylose by XylT competitively, with a

K
i

of 220 μM.
Two-Component Anti-
Staphylococcus aureus
Lantibiotic Activity Produced by
Staphylococcus aureus
C55
Authors: Navaratna Maduwe A. D. B. , Sahl Hans-Georg , Tagg John R. 
Published: December 2019

ABSTRACT

Staphylococcus aureus
C55 was shown to produce bacteriocin activity comprising three distinct peptide components, termed staphylococcins C55α, C55β, and C55γ. The three peptides were purified to homogeneity by a simple four-step purification procedure that consisted of ammonium sulfate precipitation followed by XAD-2 and reversed-phase (C
8
and C
18
) chromatography. The yield following C
8
chromatography was about 86%, with a more-than-300-fold increase in specific activity. When combined in approximately equimolar amounts, staphylococcins C55α and C55β acted synergistically to kill
S. aureus
or
Micrococcus luteus
but not
S. epidermidis
strains. The N-terminal amino acid sequences of all three peptides were obtained and staphylococcins C55α and C55β were shown to be lanthionine-containing (lantibiotic) molecules with molecular weights of 3,339 and 2,993, respectively. The C55γ peptide did not appear to be a lantibiotic, nor did it augment the inhibitory activities of staphylococcin C55α and/or C55β. Plasmids of 2.5 and 32.0 kb are present in strain C55, and following growth of this strain at elevated temperature (42°C), a large proportion of the progeny failed to produce strong bacteriocin activity and also lost the 32.0-kb plasmid. Protoplast transformation of these bacteria with purified 32-kb plasmid DNA regenerates the ability to produce the strong bacteriocin activity.
Characterization of the Prolyl Dipeptidyl Peptidase Gene (
dppIV
) from the Koji Mold
Aspergillus oryzae
Authors: Doumas Agnès , van den Broek Peter , Affolter Michael , Monod Michel 
Published: December 2019

ABSTRACT

The koji mold
Aspergillus oryzae
secretes a prolyl dipeptidyl peptidase (DPPIV) when the fungus is cultivated in a medium containing wheat gluten as the sole nitrogen and carbon source (MMWG). We cloned and sequenced the DPPIV gene from an
A. oryzae
library by using the
A. fumigatus dppIV
gene as a probe. Reverse transcriptase PCR experiments showed that the
A. oryzae dppIV
gene consists of two exons, the first of which is only 6 bp long. The gene encodes an 87.2-kDa polypeptide chain which is secreted into the medium as a 95-kDa glycoprotein. Introduction of this gene into
A. oryzae
leads to overexpression of prolyl dipeptidyl peptidase activity, while disruption of the gene abolishes all prolyl dipeptidyl peptidase activity in MMWG. The
dppIV
null mutants did not exhibit any change in phenotype other than the absence of prolyl dipeptidyl peptidase activity, suggesting that this activity is not essential. This loss of activity diminished the number of dipeptides and increased the number of larger peptides present in the MMWG culture broth. These effects were reversed by the addition of purified, recombinant DPPIV from the methylotrophic yeast expression vector
Pichia pastoris
. Our results suggest that the DPPIV enzyme may be of importance in industrial hydrolysis of what gluten-based substrates, which are rich in Pro residues.
Metabolism of Plant Polysaccharides by
Leucoagaricus gongylophorus
, the Symbiotic Fungus of the Leaf-Cutting Ant
Atta sexdens
L
Authors: Gomes De Siqueira Célia , Bacci Maurício , Pagnocca Fernando Carlos , Bueno Odair Correa , Hebling Maria JoséAparecida 
Published: December 2019

ABSTRACT

Atta sexdens
L. ants feed on the fungus they cultivate on cut leaves inside their nests. The fungus,
Leucoagaricus gongylophorus
, metabolizes plant polysaccharides, such as xylan, starch, pectin, and cellulose, mediating assimilation of these compounds by the ants. This metabolic integration may be an important part of the ant-fungus symbiosis, and it involves primarily xylan and starch, both of which support rapid fungal growth. Cellulose seems to be less important for symbiont nutrition, since it is poorly degraded and assimilated by the fungus. Pectin is rapidly degraded but slowly assimilated by
L. gongylophorus
, and its degradation may occur so that the fungus can more easily access other polysaccharides in the leaves.
Sinorhizobium fredii
and
Sinorhizobium meliloti
Produce Structurally Conserved Lipopolysaccharides and Strain-Specific K Antigens
Authors: Reuhs Bradley L. , Geller Daniel P. , Kim John S. , Fox Jennifer E. , Kolli V. S. Kumar , Pueppke Steven G. 
Published: December 2019

ABSTRACT

Lipopolysaccharides (LPS) and capsular polysaccharides (K antigens) may influence the interaction of rhizobia with their specific hosts; therefore, we conducted a comparative analysis of
Sinorhizobium fredii
and
Sinorhizobium meliloti
, which are genetically related, yet symbiotically distinct, nitrogen-fixing microsymbionts of legumes. We found that both species typically produce strain-specific K antigens that consist of 3-deoxy-
d
-
manno
-2-octulosonic acid (Kdo), or other 1-carboxy-2-keto-3-deoxy sugars (such as sialic acid), and hexoses. The K antigens of each strain are distinguished by glycosyl composition, anomeric configuration, acetylation, and molecular weight distribution. One consistent difference between the K antigens of
S. fredii
and those of
S. meliloti
is the presence of
N
-acetyl groups in the polysaccharides of the latter. In contrast to the K antigens, the LPS of
Sinorhizobium
spp. are major common antigens. Rough (R) LPS is the predominant form of LPS produced by cultured cells, and some strains release almost no detectable smooth (S) LPS upon extraction.
Sinorhizobium
spp. are delineated into two major RLPS core serogroups, which do not correspond to species (i.e., host range). The O antigens of the SLPS, when present, have similar degrees of polymerization and appear to be structurally conserved throughout the genus. Interestingly, one strain was found to be distinct from all others:
S. fredii
HH303 produces a unique K antigen, which contains galacturonic acid and rhamnose, and the RLPS did not fall into either of the RLPS core serogroups. The results of this study indicate that the conserved S- and RLPS of
Sinorhizobium
spp. lack the structural information necessary to influence host specificity, whereas the variable K antigens may affect strain-cultivar interactions.
Characterization ofcryGenes in a MexicanBacillus thuringiensisStrain Collection
Authors: Bravo Alejandra , Sarabia Sergio , Lopez Lorena , Ontiveros Hernesto , Abarca Carolina , Ortiz Anabel , Ortiz Miriam , Lina Laura , Villalobos Francisco J. , Peña Guadalupe , Nuñez-Valdez María-Eugenia , Soberón Mario , Quintero Rodolfo 
Published: December 2019

ABSTRACTMexico is located in a transition zone between the Nearctic and Neotropical biogeographical regions and contains a rich and unique biodiversity. A total of 496Bacillus thuringiensisstrains were isolated from 503 soil samples collected from the five macroregions of the country. The characterization of the strain collection provided useful information on the ecological patterns of distribution ofB. thuringiensisand opportunities for the selection of strains to develop novel bioinsecticidal products. The analysis of the strains was based on multiplex PCR with novel general and specific primers that could detect thecry1,cry3,cry5,cry7,cry8,cry9,cry11,cry12,cry13,cry14,cry21, andcytgenes. The proteins belonging to the Cry1 and Cry9 groups are toxic for lepidopteran insects. The Cry3, Cry7, and Cry8 proteins are active against coleopteran insects. The Cry5, Cry12, Cry13, and Cry14 proteins are nematocidal. The Cry11, Cry21, and Cyt proteins are toxic for dipteran insects. Six pairs of general primers are used in this method. Strains for which unique PCR product profiles were obtained with the general primers were further characterized by additional PCRs with specific primers. Strains containingcry1genes were the most abundant in our collection (49.5%). Thirty-three differentcry1-type profiles were identified.B. thuringiensisstrains harboringcry3genes represented 21.5% of the strains, and 7.9% of the strains containedcry11andcytgenes.cry7,cry8, andcry9genes were found in 0.6, 2.4, and 2.6% of the strains, respectively. No strains carryingcry5,cry12,cry13,cry14, orcry21genes were found. Finally, 14% of the strains did not give any PCR product and did not react with any polyclonal antisera. Our results indicate the presence of strains that may harbor potentially novel Cry proteins as well as strains with combinations of less frequently observedcrygenes.
Changes in Bacterial Species Composition in Enrichment Cultures with Various Dilutions of Inoculum as Monitored by Denaturing Gradient Gel Electrophoresis
Authors: Jackson Colin R. , Roden Eric E. , Churchill Perry F. 
Published: December 2019

ABSTRACT
Denaturing gradient gel electrophoresis revealed changes in the bacterial species obtained from enrichment cultures with different inoculum dilutions. This inoculum dilution enrichment approach may facilitate the detection and isolation of a greater number of bacterial species than traditional enrichment techniques.
Transfer of Streptomycin Biosynthesis Gene Clusters within Streptomycetes Isolated from Soil
Authors: Egan Sharon , Wiener Pamela , Kallifidas Dimitrios , Wellington Elizabeth M. H. 
Published: December 2019

ABSTRACT

Streptomyces
strains isolated from soil were found to possess various numbers of genes from the streptomycin biosynthesis cluster. The strains missing genes from the cluster also lacked the ability to produce streptomycin. Two of the isolates which contain only part of the cluster are apparently recipients of a gene transfer event. The implications for the role of gene transfer in antibiotic evolution are discussed.
Enzymatic Combustion of Aromatic and Aliphatic Compounds by Manganese Peroxidase from
Nematoloma frowardii
Authors: Hofrichter Martin , Scheibner Katrin , Schneegaß Ivonne , Fritsche Wolfgang 
Published: December 2019

ABSTRACT

The direct involvement of manganese peroxidase (MnP) in the mineralization of natural and xenobiotic compounds was evaluated. A broad spectrum of aromatic substances were partially mineralized by the MnP system of the white rot fungus
Nematoloma frowardii
. The cell-free MnP system partially converted several aromatic compounds, including [U-
14
C]pentachlorophenol ([U-
14
C]PCP), [U-
14
C]catechol, [U-
14
C]tyrosine, [U-
14
C]tryptophan, [4,5,9,10-
14
C]pyrene, and [ring U-
14
C]2-amino-4,6-dinitrotoluene ([
14
C]2-AmDNT), to
14
CO
2
. Mineralization was dependent on the ratio of MnP activity to concentration of reduced glutathione (thiol-mediated oxidation), a finding which was demonstrated by using [
14
C]2-AmDNT as an example. At [
14
C]2-AmDNT concentrations ranging from 2 to 120 μM, the amount of released
14
CO
2
was directly proportional to the concentration of [
14
C]2-AmDNT. The formation of highly polar products was also observed with [
14
C]2-AmDNT and [U-
14
C]PCP; these products were probably low-molecular-weight carboxylic acids. Among the aliphatic compounds tested, glyoxalate was mineralized to the greatest extent. Eighty-six percent of the
14
COOH-glyoxalate and 9% of the
14
CHO-glyoxalate were converted to
14
CO
2
, indicating that decarboxylation reactions may be the final step in MnP-catalyzed mineralization. The extracellular enzymatic combustion catalyzed by MnP could represent an important pathway for the formation of carbon dioxide from recalcitrant xenobiotic compounds and may also have general significance in the overall biodegradation of resistant natural macromolecules, such as lignins and humic substances.
Significance of Viral Lysis and Flagellate Grazing as Factors Controlling Bacterioplankton Production in a Eutrophic Lake
Authors: Weinbauer Markus G. , Höfle Manfred G. 
Published: December 2019

ABSTRACT
The effects of viral lysis and heterotrophic nanoflagellate (HNF) grazing on bacterial mortality were estimated in a eutrophic lake (Lake Plußsee in northern Germany) which was separated by a steep temperature and oxygen gradient into a warm and oxic epilimnion and a cold and anoxic hypolimnion. Two transmission electron microscopy-based methods (whole-cell examination and thin sections) were used to determine the frequency of visibly infected cells, and a model was used to estimate bacterial mortality due to viral lysis. Examination of thin sections also showed that between 20.2 and 29.2% (average, 26.1%) of the bacterial cells were empty (ghosts) and thus could not contribute to viral production. The most important finding was that the mechanism for regulating bacterial production shifted with depth from grazing control in the epilimnion to control due to viral lysis in the hypolimnion. We estimated that in the epilimnion viral lysis accounted on average for 8.4 to 41.8% of the summed mortality (calculated by determining the sum of the mortalities due to lysis and grazing), compared to 51.3 to 91.0% of the summed mortality in the metalimninon and 88.5 to 94.2% of the summed mortality in the hypolimnion. Estimates of summed mortality values indicated that bacterial production was controlled completely or almost completely in the epilimnion (summed mortality, 66.6 to 128.5%) and the hypolimnion (summed mortality, 43.4 to 103.3%), whereas in the metalimnion viral lysis and HNF grazing were not sufficient to control bacterial production (summed mortality, 22.4 to 56.7%). The estimated contribution of organic matter released by viral lysis of cells into the pool of dissolved organic matter (DOM) was low; however, since cell lysis products are very likely labile compared to the bulk DOM, they might stimulate bacterial production. The high mortality of bacterioplankton due to viral lysis in anoxic water indicates that a significant portion of bacterial production in the metalimnion and hypolimnion is cycled in the bacterium-virus-DOM loop. This finding has major implications for the fate and cycling of organic nutrients in lakes.