bluuebunny/crossref_metadata_2025_split huggingface dataset:
Partial Purification and Characterization of
Bacillus thuringiensis
Cry1A Toxin Receptor A from
Heliothis virescens
and Cloning of the Corresponding cDNA
Authors: Oltean Daniela I. , Pullikuth Ashok K. , Lee Hyun-Ku , Gill Sarjeet S. 
Published: December 2019

ABSTRACT

Although extensively studied, the mechanism of action of insecticidal
Bacillus thuringiensis
Cry toxins remains elusive and requires further elucidation. Toxin receptors in the brush border membrane demand particular attention as they presumably initiate the cascade of events leading to insect mortality after toxin activation. The 170-kDa Cry1Ac toxin-binding aminopeptidase from the tobacco budworm (
Heliothis virescens
) was partially purified, and its corresponding cDNA was cloned. The cDNA encodes a protein with a putative glycosyl phosphatidylinositol anchor and a polythreonine stretch clustered near the C terminus with predicted O-glycosylation. Partial purification of the 170-kDa aminopeptidase also resulted in isolation of a 130-kDa protein that was immunologically identical to the 170-kDa protein, and the two proteins had identical N termini. These proteins were glycosylated, as suggested by soybean agglutinin lectin blot results. Cry1Ac toxin affinity data for the two proteins indicated that the 130-kDa protein had a higher affinity than the 170-kDa protein. The data suggest that posttranslational modifications can have a significant effect on Cry1A toxin interactions with specific insect midgut proteins.
Characterization of an Operon Encoding Two
c
-Type Cytochromes, an

aa
3

-Type Cytochrome Oxidase, and Rusticyanin in
Thiobacillus ferrooxidans
ATCC 33020
Authors: Appia-Ayme Corinne , Guiliani Nicolas , Ratouchniak Jeanine , Bonnefoy Violaine 
Published: December 2019

ABSTRACT

Despite the importance of
Thiobacillus ferrooxidans
in bioremediation and bioleaching, little is known about the genes encoding electron transfer proteins implicated in its energetic metabolism. This paper reports the sequences of the four
cox
genes encoding the subunits of an
aa
3
-type cytochrome
c
oxidase. These genes are in a locus containing four other genes:
cyc2
, which encodes a high-molecular-weight cytochrome
c
;
cyc1
, which encodes a
c
4
-type cytochrome (
c
552
); open reading frame 1, which encodes a putative periplasmic protein of unknown function; and
rus
, which encodes rusticyanin. The results of Northern and reverse transcription-PCR analyses indicated that these eight genes are cotranscribed. Two transcriptional start sites were identified for this operon. Upstream from each of the start sites was a ς70-type promoter recognized in
Escherichia coli
. While transcription in sulfur-grown
T. ferrooxidans
cells was detected from the two promoters, transcription in ferrous-iron-grown
T. ferrooxidans
cells was detected only from the downstream promoter. The cotranscription of seven genes encoding redox proteins suggests that all these proteins are involved in the same electron transfer chain; a model taking into account the biochemistry and the genetic data is discussed.
Transposon-Induced Mutations in Two Loci of
Listeria monocytogenes
Serotype 1/2a Result in Phage Resistance and Lack of
N
-Acetylglucosamine in the Teichoic Acid of the Cell Wall
Authors: Tran Huyen L. , Fiedler F. , Hodgson D. A. , Kathariou S. 
Published: December 2019

ABSTRACT

Teichoic acid-associated
N
-acetylglucosamine and rhamnose have been shown to serve as phage receptors in
Listeria monocytogenes
serotype 1/2a. We generated and characterized two single-copy Tn
916
ΔE mutants which were resistant to phage A118 and several other serotype 1/2a-specific phages. In one mutant the insertion was immediately upstream of the recently identified
ptsHI
locus, which encodes two proteins of the phosphoenolpyruvate-dependent carbohydrate uptake system, whereas in the other the insertion was immediately upstream of an operon whose most distal gene was
clpC
, involved in stress responses and virulence. Transduction experiments confirmed the association of the phage-resistant phenotype of these mutants with the transposon insertion. Phage A118 resistance of the mutants could be attributed to inability of the phage to adsorb onto the mutant cells, and biochemical analysis of cell wall composition showed that the teichoic acids of both mutants were deficient in
N
-acetylglucosamine. Rhamnose and other teichoic acid and cell wall components were not affected.
Distribution of Tetracycline Resistance Genes and Transposons among Phylloplane Bacteria in Michigan Apple Orchards
Authors: Schnabel Elise L. , Jones Alan L. 
Published: December 2019

ABSTRACT

The extent and nature of tetracycline resistance in bacterial populations of two apple orchards with no or a limited history of oxytetracycline usage were assessed. Tetracycline-resistant (Tc
r
) bacteria were mostly gram negative and represented from 0 to 47% of the total bacterial population on blossoms and leaves (versus 26 to 84% for streptomycin-resistant bacteria). A total of 87 isolates were screened for the presence of specific Tc
r
determinants. Tc
r
was determined to be due to the presence of Tet B in
Pantoea agglomerans
and other members of the family
Enterobacteriacae
and Tet A, Tet C, or Tet G in most
Pseudomonas
isolates. The cause of Tc
r
was not identified in 16% of the isolates studied. The Tc
r
genes were almost always found on large plasmids which also carried the streptomycin resistance transposon Tn
5393
. Transposable elements with Tc
r
determinants were detected by entrapment following introduction into
Escherichia coli
. Tet B was found within Tn
10
. Two of eighteen Tet B-containing isolates had an insertion sequence within Tn
10
; one had IS
911
located within IS
10
-R and one had Tn
1000
located upstream of Tet B. Tet A was found within a novel variant of Tn
1721
, named Tn
1720
, which lacks the left-end
orfI
of Tn
1721
. Tet C was located within a 19-kb transposon, Tn
1404
, with transposition genes similar to those of Tn
501
, streptomycin (
aadA2
) and sulfonamide (
sulI
) resistance genes within an integron, Tet C flanked by direct repeats of IS
26
, and four open reading frames, one of which may encode a sulfate permease. Two variants of Tet G with 92% sequence identity were detected.
PCR Amplification of the Gene
acmA
Differentiates
Lactococcus lactis
subsp.
lactis
and
L. lactis
subsp.
cremoris
Authors: Garde Sonia , Babin Mar , Gaya Pilar , Nuñez Manuel , Medina Margarita 
Published: December 2019

ABSTRACT

The occurrence of the
acmA
gene, encoding the lactococcal
N
-acetylmuramidase in new lactococcal isolates from raw milk cheeses, has been determined. Isolates were genotypically identified to the subspecies level with a PCR technique. On the basis of PCR amplification of the
acmA
gene, the presence or absence of an additional amplicon of approximately 700 bp correlated with
Lactococcus lactis
subspecies.
L. lactis
subsp.
lactis
exhibits both the expected 1,131-bp product and the additional amplicon, whereas
L. lactis
subsp.
cremoris
exhibits a single 1,131-bp fragment.
Influence of Different Electron Donors and Acceptors on Dehalorespiration of Tetrachloroethene by
Desulfitobacterium frappieri
TCE1
Authors: Gerritse Jan , Drzyzga Oliver , Kloetstra Geert , Keijmel Mischa , Wiersum Luit P. , Hutson Roger , Collins Matthew D. , Gottschal Jan C. 
Published: December 2019

ABSTRACT

Strain TCE1, a strictly anaerobic bacterium that can grow by reductive dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE), was isolated by selective enrichment from a PCE-dechlorinating chemostat mixed culture. Strain TCE1 is a gram-positive, motile, curved rod-shaped organism that is 2 to 4 by 0.6 to 0.8 μm and has approximately six lateral flagella. The pH and temperature optima for growth are 7.2 and 35°C, respectively. On the basis of a comparative 16S rRNA sequence analysis, this bacterium was identified as a new strain of
Desulfitobacterium frappieri
, because it exhibited 99.7% relatedness to the
D. frappieri
type strain, strain PCP-1. Growth with H
2
, formate,
l
-lactate, butyrate, crotonate, or ethanol as the electron donor depends on the availability of an external electron acceptor. Pyruvate and serine can also be used fermentatively. Electron donors (except formate and H
2
) are oxidized to acetate and CO
2
. When
l
-lactate is the growth substrate, strain TCE1 can use the following electron acceptors: PCE and TCE (to produce
cis
-1,2-dichloroethene), sulfite and thiosulfate (to produce sulfide), nitrate (to produce nitrite), and fumarate (to produce succinate). Strain TCE1 is not able to reductively dechlorinate 3-chloro-4-hydroxyphenylacetate. The growth yields of the newly isolated bacterium when PCE is the electron acceptor are similar to those obtained for other dehalorespiring anaerobes (e.g.,
Desulfitobacterium
sp. strain PCE1 and
Desulfitobacterium hafniense
) and the maximum specific reductive dechlorination rates are 4 to 16 times higher (up to 1.4 μmol of chloride released · min
−1
· mg of protein
−1
). Dechlorination of PCE and TCE is an inducible process. In PCE-limited chemostat cultures of strain TCE1, dechlorination is strongly inhibited by sulfite but not by other alternative electron acceptors, such as fumarate or nitrate.
Dynamic Interaction of
Trichoderma reesei
Cellobiohydrolases Cel6A and Cel7A and Cellulose at Equilibrium and during Hydrolysis
Authors: Palonen Hetti , Tenkanen Maija , Linder Markus 
Published: December 2019

ABSTRACT

The binding of cellobiohydrolases to cellulose is a crucial initial step in cellulose hydrolysis. In the search for a detailed understanding of the function of cellobiohydrolases, much information concerning how the enzymes and their constituent catalytic and cellulose-binding domains interact with cellulose and with each other and how binding changes during hydrolysis is still needed. In this study we used tritium labeling by reductive methylation to monitor binding of the two
Trichoderma reesei
cellobiohydrolases, Cel6A and Cel7A (formerly CBHII and CBHI), and their catalytic domains. Measuring hydrolysis by high-performance liquid chromatography and measuring binding by scintillation counting allowed us to correlate activity and binding as a function of the extent of degradation. These experiments showed that the density of bound protein increased with both Cel6A and Cel7A as hydrolysis proceeded, in such a way that the adsorption points moved off the initial binding isotherms. We also compared the affinities of the cellulose-binding domains and the catalytic domains to the affinities of the intact proteins and found that in each case the affinity of the enzyme was determined by the linkage between the catalytic and cellulose-binding domains. Desorption of Cel6A by dilution of the sample showed hysteresis (60 to 70% reversible); in contrast, desorption of Cel7A did not show hysteresis and was more than 90% reversible. These findings showed that the two enzymes differ with respect to the reversibility of binding.
cis
-Chlorobenzene Dihydrodiol Dehydrogenase (TcbB) from
Pseudomonas
sp. Strain P51, Expressed in
Escherichia coli
DH5α(pTCB149), Catalyzes Enantioselective Dehydrogenase Reactions
Authors: Raschke Henning , Fleischmann Thomas , Van Der Meer Jan Roelof , Kohler Hans-Peter E. 
Published: December 2019

ABSTRACT

cis
-Chlorobenzene dihydrodiol dehydrogenase (CDD) from
Pseudomonas
sp. strain P51, cloned into
Escherichia coli
DH5α(pTCB149) was able to oxidize
cis
-dihydrodihydroxy derivatives (
cis
-dihydrodiols) of dihydronaphthalene, indene, and four
para
-substituted toluenes to the corresponding catechols. During the incubation of a nonracemic mixture of
cis
-1,2-indandiol, only the (+)-
cis
-(1
R
,2
S
) enantiomer was oxidized; the (−)-
cis
-(
S
,2
R
) enantiomer remained unchanged. CDD oxidized both enantiomers of
cis
-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene, but oxidation of the (+)-
cis
-(1
S
,2
R
) enantiomer was delayed until the (−)-
cis
-(1
R
,2
S
) enantiomer was completely depleted. When incubated with nonracemic mixtures of
para
-substituted
cis
-toluene dihydrodiols, CDD always oxidized the major enantiomer at a higher rate than the minor enantiomer. When incubated with racemic 1-indanol, CDD enantioselectively transformed the (+)-(1
S
) enantiomer to 1-indanone. This stereoselective transformation shows that CDD also acted as an alcohol dehydrogenase. Additionally, CDD was able to oxidize (+)-
cis
-(1
R
,2
S
)-dihydroxy-1,2-dihydronaphthalene, (+)-
cis
-monochlorobiphenyl dihydrodiols, and (+)-
cis
-toluene dihydrodiol to the corresponding catechols.
A Unique Chitinase with Dual Active Sites and Triple Substrate Binding Sites from the Hyperthermophilic Archaeon
Pyrococcus kodakaraensis
KOD1
Authors: Tanaka Takeshi , Fujiwara Shinsuke , Nishikori Shingo , Fukui Toshiaki , Takagi Masahiro , Imanaka Tadayuki 
Published: December 2019

ABSTRACT

We have found that the hyperthermophilic archaeon
Pyrococcus kodakaraensis
KOD1 produces an extracellular chitinase. The gene encoding the chitinase (
chiA
) was cloned and sequenced. The
chiA
gene was found to be composed of 3,645 nucleotides, encoding a protein (1,215 amino acids) with a molecular mass of 134,259 Da, which is the largest among known chitinases. Sequence analysis indicates that ChiA is divided into two distinct regions with respective active sites. The N-terminal and C-terminal regions show sequence similarity with chitinase A1 from
Bacillus circulans
WL-12 and chitinase from
Streptomyces erythraeus
(ATCC 11635), respectively. Furthermore, ChiA possesses unique chitin binding domains (CBDs) (CBD1, CBD2, and CBD3) which show sequence similarity with cellulose binding domains of various cellulases. CBD1 was classified into the group of family V type cellulose binding domains. In contrast, CBD2 and CBD3 were classified into that of the family II type.
chiA
was expressed in
Escherichia coli
cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for chitinase activity were found to be 85°C and 5.0, respectively. Results of thin-layer chromatography analysis and activity measurements with fluorescent substrates suggest that the enzyme is an endo-type enzyme which produces a chitobiose as a major end product. Various deletion mutants were constructed, and analyses of their enzyme characteristics revealed that both the N-terminal and C-terminal halves are independently functional as chitinases and that CBDs play an important role in insoluble chitin binding and hydrolysis. Deletion mutants which contain the C-terminal half showed higher thermostability than did N-terminal-half mutants and wild-type ChiA.
Mitochondrial Function in Cell Wall Glycoprotein Synthesis in
Saccharomyces cerevisiae
NCYC 625 (Wild Type) and [
rho
0
] Mutants
Authors: Iung Annie Rakotoarivony , Coulon Joël , Kiss Ferenc , Ekome Jacques Ngondi , Vallner Judit , Bonaly Roger 
Published: December 2019

ABSTRACT

We studied phosphopeptidomannans (PPMs) of two
Saccharomyces cerevisiae
NCYC 625 strains (
S. diastaticus
): a wild type strain grown aerobically, anaerobically, and in the presence of antimycin and a [
rho
0
] mutant grown aerobically and anaerobically. The aerobic wild-type cultures were highly flocculent, but all others were weakly flocculent. Ligands implicated in flocculation of mutants or antimycin-treated cells were not aggregated as much by concanavalin A as were those of the wild type. The [
rho
0
] mutants and antimycin-treated cells differ from the wild type in PPM composition and invertase, acid phosphatase, and glucoamylase activities. PPMs extracted from different cells differ in the protein but not in the glycosidic moiety. The PPMs were less stable in mitochondrion-deficient cells than in wild-type cells grown aerobically, and this difference may be attributable to defective mitochondrial function during cell wall synthesis. The reduced flocculation of cells grown in the presence of antimycin, under anaerobiosis, or carrying a [
rho
0
] mutation may be the consequence of alterations of PPM structures which are the ligands of lectins, both involved in this cell-cell recognition phenomenon. These respiratory chain alterations also affect peripheral, biologically active glycoproteins such as extracellular enzymes and peripheral PPMs.
Isolation and Characterization of the Epoxide Hydrolase-Encoding Gene from
Xanthophyllomyces dendrorhous
Authors: Visser Hans , de Bont Jan A. M. , Verdoes Jan C. 
Published: December 2019

ABSTRACT

The epoxide hydrolase (EH)-encoding gene (
EPH1
) from the basidiomycetous yeast
Xanthophyllomyces dendrorhous
was isolated. The genomic sequence has a 1,236-bp open reading frame which is interrupted by eight introns that encode a 411-amino-acid polypeptide with a calculated molecular mass of 46.2 kDa. The amino acid sequence is similar to that of microsomal EH and belongs to the α/β hydrolase fold family. The
EPH1
gene was not essential for growth of
X. dendrorhous
in rich medium under laboratory conditions. The Eph1-encoding cDNA was functionally expressed in
Escherichia coli
. A sixfold increase in specific activity was observed when we used resting cells rather than
X. dendrorhous
. The epoxides 1,2-epoxyhexane and 1-methylcyclohexene oxide were substrates for both native and recombinant Eph1. Isolation and characterization of the
X. dendrorhous
EH-encoding gene are essential steps in developing a yeast EH-based epoxide biotransformation system.
Novel Interaction between Laccase and Cellobiose Dehydrogenase during Pigment Synthesis in the White Rot Fungus
Pycnoporus cinnabarinus
Authors: Temp Ulrike , Eggert Claudia 
Published: December 2019

ABSTRACT

When glucose is the carbon source, the white rot fungus
Pycnoporus cinnabarinus
produces a characteristic red pigment, cinnabarinic acid, which is formed by laccase-catalyzed oxidation of the precursor 3-hydroxyanthranilic acid. When
P. cinnabarinus
was grown on media containing cellobiose or cellulose as the carbon source, the amount of cinnabarinic acid that accumulated was reduced or, in the case of cellulose, no cinnabarinic acid accumulated. Cellobiose-dependent quinone reducing enzymes, the cellobiose dehydrogenases (CDHs), inhibited the redox interaction between laccase and 3-hydroxyanthranilic acid. Two distinct proteins were purified from cellulose-grown cultures of
P. cinnabarinus
; these proteins were designated CDH I and CDH II. CDH I and CDH II were both monomeric proteins and had apparent molecular weights of about 81,000 and 101,000, respectively, as determined by both gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pI values were approximately 5.9 for CDH I and 3.8 for CDH II. Both CDHs used several known CDH substrates as electron acceptors and specifically adsorbed to cellulose. Only CDH II could reduce cytochrome
c
. The optimum pH values for CDH I and CDH II were 5.5 and 4.5, respectively. In in vitro experiments, both enzymes inhibited laccase-mediated formation of cinnabarinic acid. Oxidation intermediates of 3-hydroxyanthranilic acid served as endogenous electron acceptors for the two CDHs from
P. cinnabarinus
. These results demonstrated that in the presence of a suitable cellulose-derived electron donor, CDHs can regenerate fungal metabolites oxidized by laccase, and they also supported the hypothesis that CDHs act as links between cellulolytic and ligninolytic pathways.
Composition and Enzymatic Activity of the Extracellular Matrix Secreted by Germlings ofBotrytis cinerea
Authors: Doss Robert P. 
Published: December 2019

ABSTRACTGermlings ofBotrytis cinerea, an important fungal pathogen of plants, produce an extracellular matrix (ECM), or ensheathing film, that serves, in part, in their attachment (R. P. Doss, et al., Appl. Environ. Microbiol. 61:260–265, 1995). The composition of this film has been ascertained by using samples obtained by growing germlings on a glass surface, removing the fungal mycelium by vigorous washing, and collecting the tightly attached film by scraping the substratum with a razor blade. Slightly over half of the dry weight of the ECM was found to be carbohydrates (about 20%), proteins (about 28%), and lipids (about 6%). Hydrolysis of the carbohydrate portion of the ECM revealed that glucose was the most prominent monosaccharide present, comprising about 60% of the total monosaccharides. Also present were mannose (about 35%) andmyo-inositol (about 5%). The proteinaceous fraction of the ECM was made up of a number of polypeptides separable by polyacrylamide gel electrophoresis. The lipid fraction of the ECM, analyzed by thin-layer chromatography, was made up of several simple lipid components, including free fatty acid, mono- and triacylglycerol, wax ester, fatty alcohol, and several unidentified components. No complex lipids were detected. Isolated ECM exhibited polygalacturonase and laccase activity and was able to catalyze the hydrolysis ofp-nitrophenyl butyrate, a model substrate for assessing cutinase activity. Cellulase, pectin lyase, and pectin methyl esterase activities were noted with both heated and unheated ECM preparations. Proteinase activity was not detected.
Green Fluorescent Protein as a Noninvasive Stress Probe in Resting
Escherichia coli
Cells
Authors: Cha Hyung Joon , Srivastava Ranjan , Vakharia Vikram N. , Rao Govind , Bentley William E. 
Published: December 2019

ABSTRACT

We constructed and characterized three stress probe plasmids which utilize a green fluorescent protein as a noninvasive reporter in order to elucidate
Escherichia coli
cellular stress responses in quiescent or resting cells. Cellular stress levels were easily detected by fusing three heat shock stress protein promoter elements, those of the heat shock transcription factor ς
32
, the protease subunit ClpB, and the chaperone DnaK, to the reporter gene

gfp
uv

. When perturbed by a chemical or physical stress (such as a heat shock, nutrient [amino acid] limitation, or addition of IPTG [isopropyl-β-
d
-thiogalactopyranoside], acetic acid, ethanol, phenol, antifoam, or salt [osmotic shock]), the
E. coli
cells produced GFPuv, which was easily detected within the cells as emitted green fluorescence. Temporal and amplitudinal mapping of the responses was performed, and the results revealed regions where quantitative delineation of cell stress was afforded.