bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural Characterization of the Symbiotically Important Low-Molecular-Weight Succinoglycan of
Sinorhizobium meliloti
Authors: Wang Lai-Xi , Wang Ying , Pellock Brett , Walker Graham C. 
Published: December 2019

ABSTRACT

The production of succinoglycan by
Sinorhizobium meliloti
Rm1021 is required for successful nodule invasion by the bacterium of its host plant, alfalfa. Rm1021 produces succinoglycan, an acidic exopolysaccharide composed of an octasaccharide repeating unit modified with acetyl, succinyl, and pyruvyl moieties, in both low- and high-molecular-weight forms. Low-molecular-weight (LMW) succinoglycan, previously thought to consist of monomers, trimers, and tetramers of the repeating unit, has been reported as being capable of promoting the formation of nitrogen-fixing nodules by succinoglycan-deficient derivatives of strain Rm1021. We have determined that the three size classes of LMW succinoglycan species are in fact monomers, dimers, and trimers of the repeating unit and that the trimer is the species active in promoting nodule invasion. A detailed structural analysis of the components of LMW succinoglycan by using various chromatographic techniques, along with nuclear magnetic resonance analyses, has revealed that there is considerable heterogeneity within the LMW succinoglycan oligomers in terms of noncarbohydrate substitutions, and we have determined the structural basis of this heterogeneity.
Cloning and Expression of
Mycobacterium tuberculosis
and
Mycobacterium leprae
Dihydropteroate Synthase in
Escherichia coli
Authors: Nopponpunth Vanida , Sirawaraporn Worachart , Greene Patricia J. , Santi Daniel V. 
Published: December 2019

ABSTRACT

The genes for dihydropteroate synthase of
Mycobacterium tuberculosis
and
Mycobacterium leprae
were isolated by hybridization with probes amplified from the genomic DNA libraries. DNA sequencing revealed an open reading frame of 840 bp encoding a protein of 280 amino acids for
M. tuberculosis
dihydropteroate synthase and an open reading frame of 852 bp encoding a protein of 284 amino acids for
M. leprae
dihydropteroate synthase. The dihydropteroate synthases were expressed under control of the T5 promoter in a dihydropteroate synthase-deficient strain of
Escherichia coli
. Using three chromatography steps, we purified both
M. tuberculosis
and
M. leprae
dihydropteroate synthases to >98% homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed molecular masses of 29 kDa for
M. tuberculosis
dihydropteroate synthase and 30 kDa for
M. leprae
dihydropteroate synthase. Gel filtration of both enzymes showed a molecular mass of ca. 60 kDa, indicating that the native enzymes exist as dimers of two identical subunits. Steady-state kinetic parameters for dihydropteroate synthases from both
M. tuberculosis
and
M. leprae
were determined. Representative sulfonamides and dapsone were potent inhibitors of the mycobacterial dihydropteroate synthases, but the antimycobacterial agent
p
-aminosalicylate, a putative dihydropteroate synthase inhibitor, was a poor inhibitor of the enzymes.
CzcD Is a Heavy Metal Ion Transporter Involved in Regulation of Heavy Metal Resistance in
Ralstonia
sp. Strain CH34
Authors: Anton Andreas , Große Cornelia , Reißmann Jana , Pribyl Thomas , Nies Dietrich H. 
Published: December 2019

ABSTRACT

The Czc system of
Ralstonia
sp. strain CH34 mediates resistance to cobalt, zinc, and cadmium through ion efflux catalyzed by the CzcCB
2
A cation-proton antiporter. The CzcD protein is involved in the regulation of the Czc system. It is a membrane-bound protein with at least four transmembrane α-helices and is a member of a subfamily of the cation diffusion facilitator (CDF) protein family, which occurs in all three domains of life. The deletion of
czcD
in a
Ralstonia
sp. led to partially constitutive expression of the Czc system due to an increased transcription of the structural
czcCBA
genes, both in the absence and presence of inducers. The
czcD
deletion could be fully complemented in
trans
by CzcD and two other CDF proteins from
Saccharomyces cerevisiae
, ZRC1p and COT1p. All three proteins mediated a small but significant resistance to cobalt, zinc, and cadmium in
Ralstonia
, and this resistance was based on a reduced accumulation of the cations. Thus, CzcD appeared to repress the Czc system by an export of the inducing cations.
Immunochemical Characterization and Taxonomic Evaluation of the O Polysaccharides of the Lipopolysaccharides ofPseudomonas syringaeSerogroup O1 Strains
Authors: Ovod Vladimir V. , Knirel Yuriy A. , Samson Regine , Krohn Kai J. 
Published: December 2019

ABSTRACTThe O polysaccharide (OPS) of the lipopolysaccharide (LPS) ofPseudomonas syringaepv. atrofaciens IMV 7836 and some other strains that are classified in serogroup O1 was shown to be a novel linear α-d-rhamnan with the tetrasaccharide O repeat →3)-α-d-Rhap-(1→3)-α-d-Rhap-(1→2)-α-d-Rhap-(1→2)-α-d-Rhap-(1→ (chemotype 1A). The same α-d-rhamnan serves as the backbone in branched OPSs with lateral (α1→3)-linkedd-Rhap, (β1→4)-linkedd-GlcpNAc, and (α1→4)-linkedd-Fucfresidues (chemotypes 1B, 1C, and 1D, respectively). Strains of chemotype 1C demonstrated variations resulting in a decrease of the degree of substitution of the backbone 1A with the laterald-GlcNAc residue (chemotype 1C-1A), which may be described as branched regular ↔ branched irregular → linear OPS structure alterations (1C↔1C-1A → 1A). Based on serological data, chemotype 1D was suggested to undergo a 1D ↔ 1D-1A alteration, whereas chemotype 1B showed no alteration. A number of OPS backbone-specific monoclonal antibodies (MAbs), Ps(1-2)a, Ps(1-2)a1, Ps1a, Ps1a1, and Ps1a2, as well as MAbs Ps1b, Ps1c, Ps1c1, Ps1d, Ps(1-2)d, and Ps(1-2)d1specific to epitopes related to the lateral sugar substituents of the OPSs, were produced againstP. syringaeserogroup O1 strains. By using MAbs, some specific epitopes were inferred, serogroup O1 strains were serotyped in more detail, and thus, the serological classification scheme ofP. syringaewas improved. Screening with MAbs of about 800 strains representing all 56 knownP. syringaepathovars showed that the strains classified in serogroup O1 were found among 15 pathovars and the strains with the linear OPSs of chemotype 1A were found among 9 of the 15 pathovars. A possible role for the LPS ofP. syringaeand related pseudomonads as a phylogenetic marker is discussed.
A Novel Lipolytic Enzyme Located in the Outer Membrane of
Pseudomonas aeruginosa
Authors: Wilhelm Susanne , Tommassen Jan , Jaeger Karl-Erich 
Published: December 2019

ABSTRACT

A lipase-negative deletion mutant of
Pseudomonas aeruginosa
PAO1 still showed extracellular lipolytic activity toward short-chain
p
-nitrophenylesters. By screening a genomic DNA library of
P. aeruginosa
PAO1, an esterase gene,
estA
, was identified, cloned, and sequenced, revealing an open reading frame of 1,941 bp. The product of
estA
is a 69.5-kDa protein, which is probably processed by removal of an N-terminal signal peptide to yield a 67-kDa mature protein. A molecular mass of 66 kDa was determined for
35
S-labeled EstA by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The amino acid sequence of EstA indicated that the esterase is a member of a novel GDSL family of lipolytic enzymes. The
estA
gene showed high similarity to an open reading frame of unknown function located in the
trpE-trpG
region of
P. putida
and to a gene encoding an outer membrane esterase of
Salmonella typhimurium
. Amino acid sequence alignments led us to predict that this esterase is an autotransporter protein which possesses a carboxy-terminal β-barrel domain, allowing the secretion of the amino-terminal passenger domain harboring the catalytic activity. Expression of
estA
in
P. aeruginosa
and
Escherichia coli
and subsequent cell fractionation revealed that the enzyme was associated with the cellular membranes. Trypsin treatment of whole cells released a significant amount of esterase, indicating that the enzyme was located in the outer membrane with the catalytic domain exposed to the surface. To our knowledge, this esterase is unique in that it exemplifies in
P. aeruginosa
(i) the first enzyme identified in the outer membrane and (ii) the first example of a type IV secretion mechanism.
Role of CcpA in Regulation of the Central Pathways of Carbon Catabolism in
Bacillus subtilis
Authors: Tobisch Steffen , Zühlke Daniela , Bernhardt Jörg , Stülke Jörg , Hecker Michael 
Published: December 2019

ABSTRACT

The
Bacillus subtilis
two-dimensional (2D) protein index contains almost all glycolytic and tricarboxylic acid (TCA) cycle enzymes, among them the most abundant housekeeping proteins of growing cells. Therefore, a comprehensive study on the regulation of glycolysis and the TCA cycle was initiated. Whereas expression of genes encoding the upper and lower parts of glycolysis (
pgi
,
pfk
,
fbaA
, and
pykA
) is not affected by the glucose supply, there is an activation of the glycolytic
gap
gene and the
pgk
operon by glucose. This activation seems to be dependent on the global regulator CcpA, as shown by 2D polyacrylamide gel electrophoresis analysis as well as by transcriptional analysis. Furthermore, a high glucose concentration stimulates production and excretion of organic acids (overflow metabolism) in the wild type but not in the
ccpA
mutant. Finally, CcpA is involved in strong glucose repression of almost all TCA cycle genes. In addition to TCA cycle and glycolytic enzymes, the levels of many other proteins are affected by the
ccpA
mutation. Our data suggest (i) that
ccpA
mutants are unable to activate glycolysis or carbon overflow metabolism and (ii) that CcpA might be a key regulator molecule, controlling a superregulon of glucose catabolism.
Characterization of the Essential Transport Function of the AIDA-I Autotransporter and Evidence Supporting Structural Predictions
Authors: Maurer Jochen , Jose Joachim , Meyer Thomas F. 
Published: December 2019

ABSTRACT

The current model for autodisplay suggests a mechanism that allows a passenger protein to be translocated across the outer membrane by coordinate action of a C-terminal β-barrel and its preceding linking region. The passenger protein, linker, and β-barrel are together termed the autotransporter, while the linker and β-barrel are here referred to as the translocation unit (TU). We characterized the minimal TU necessary for autodisplay with the adhesin-involved-in-diffuse-adherence (AIDA-I) autotransporter. The assumed β-barrel structure at the C terminus of the AIDA-I autotransporter was studied by constructing a set of seven AIDA-I–cholera toxin B subunit fusion proteins containing various portions of AIDA-I. Surface exposure of the cholera toxin B moiety was assessed by dot blot experiments and trypsin accessibility of the chimeric proteins expressed in
Escherichia coli
JK321 or UT5600. Export of cholera toxin B strictly depended on a complete predicted β-barrel region. The absolute necessity for export of a linking region and its influence on expression as an integral part of the TU was also demonstrated. The different electrophoretic mobilities of native and denatured chimeras indicated that the proposed β-barrel resides within the C-terminal 312 amino acids of AIDA-I. Together these data provide evidence for the predicted β-barrel structure and support our formerly proposed model of membrane topology of the AIDA-I autotransporter.
Transcriptional Control of the Low-Temperature-Inducible
des
Gene, Encoding the Δ5 Desaturase of
Bacillus subtilis
Authors: Aguilar Pablo S. , Lopez Paloma , de Mendoza Diego 
Published: December 2019

ABSTRACT

The
Bacillus subtilis des
gene encodes the cold-inducible Δ5 lipid desaturase involved in the formation of unsaturated fatty acids from saturated phospholipid precursors. Here, we describe the expression pattern of the
des
gene in response to a temperature downshift from 37 to 20°C. We found that the synthesis of
des
mRNA is undetectable at 37°C but dramatically induced upon the temperature downshift. Decay characteristics of the
des
transcript as well as the in vivo decay of
B. subtilis
bulk mRNA were investigated. The results showed that the stability of the
des
transcript as well as of bulk mRNA lasted substantially longer at 20°C than at 37°C. Functional expression of
des
at 37°C was achieved by exchanging its promoter with the non-cold shock
spac
promoter. These data provide the first direct evidence that temperature-mediated control of transcription is the major mechanism regulating the mRNA levels of the
B. subtilis
desaturase. The present results also demonstrate that the only component of the desaturation system regulated by temperature is the desaturase enzyme.
Synergistic Operation of theCAR2(Ornithine Transaminase) Promoter Elements inSaccharomyces cerevisiae
Authors: Park Heui-Dong , Scott Stephanie , Rai Rajendra , Dorrington Rosemary , Cooper Terrance G. 
Published: December 2019

ABSTRACTDal82p binds to theUISALLsites of allophanate-induced genes of the allantoin-degradative pathway and functions synergistically with the GATA family Gln3p and Gat1p transcriptional activators that are responsible for nitrogen catabolite repression-sensitive gene expression.CAR2, which encodes the arginine-degradative enzyme ornithine transaminase, is not nitrogen catabolite repression sensitive, but its expression can be modestly induced by the allantoin pathway inducer. The dominant activators ofCAR2transcription have been thought to be the ArgR and Mcm1 factors, which mediate arginine-dependent induction. These observations prompted us to investigate the structure of theCAR2promoter with the objectives of determining whether other transcription factors were required forCAR2expression and, if so, of ascertaining their relative contributions toCAR2’s expression and control. We show that Rap1p binds upstream ofCAR2and plays a central role in its induced expression irrespective of whether the inducer is arginine or the allantoin pathway inducer analogue oxalurate (OXLU). Our data also explain the early report that ornithine transaminase production is induced when cells are grown with urea. OXLU induction derives from the Dal82p binding site, which is immediately downstream of the Rap1p site, and Dal82p functions synergistically with Rap1p. This synergism is unlike all other known instances of Dal82p synergism, namely, that with the GATA family transcription activators Gln3p and Gat1p, which occurs only in the presence of an inducer. The observations reported suggest thatCAR2gene expression results from strong constitutive transcriptional activation mediated by Rap1p and Dal82p being balanced by the down regulation of an equally strong transcriptional repressor, Ume6p. This balance is then tipped in the direction of expression by the presence of the inducer. The formal structure of theCAR2promoter and its operation closely follow the model proposed forCAR1.
Transcriptional Organization of the Erythromycin Biosynthetic Gene Cluster of
Saccharopolyspora erythraea
Authors: Reeves Andrew R. , English R. Samuel , Lampel J. S. , Post David A. , Vanden Boom Thomas J. 
Published: December 2019

ABSTRACT

The transcriptional organization of the erythromycin biosynthetic gene (
ery
) cluster of
Saccharopolyspora erythraea
has been examined by a variety of methods, including S1 nuclease protection assays, Northern blotting, Western blotting, and bioconversion analysis of erythromycin intermediates. The analysis was facilitated by the construction of novel mutants containing a
S. erythraea
transcriptional terminator within the
eryAI
,
eryAIII
,
eryBIII
,
eryBIV
,
eryBV
,
eryBVI
,
eryCIV
, and
eryCVI
genes and additionally by an
eryAI
−10 promoter mutant. All mutant strains demonstrated polar effects on the transcription of downstream
ery
biosynthetic genes. Our results demonstrate that the
ery
gene cluster contains four major polycistronic transcriptional units, the largest one extending approximately 35 kb from
eryAI
to
eryG
. Two overlapping polycistronic transcripts extending from
eryBIV
to
eryBVII
were identified. In addition, seven
ery
cluster promoter transcription start sites, one each beginning at
eryAI
,
eryBI
,
eryBIII
,
eryBVI
, and
eryK
and two beginning at
eryBIV
, were determined.
Chromosomal Integration of Heterologous DNA in
Escherichia coli
with Precise Removal of Markers and Replicons Used during Construction
Authors: Martinez-Morales F. , Borges A. C. , Martinez A. , Shanmugam K. T. , Ingram L. O. 
Published: December 2019

ABSTRACT

A set of vectors which facilitates the sequential integration of new functions into the
Escherichia coli
chromosome by homologous recombination has been developed. These vectors are based on plasmids described by Posfai et al. (J. Bacteriol. 179:4426–4428, 1997) which contain conditional replicons (pSC101 or R6K), a choice of three selectable markers (ampicillin, chloramphenicol, or kanamycin), and a single
FRT
site. The modified vectors contain two
FRT
sites which bracket a modified multiple cloning region for DNA insertion. After integration, a helper plasmid expressing the flippase (FLP) recombinase allows precise in vivo excision of the replicon and the marker used for selection. Sites are also available for temporary insertion of additional functions which can be subsequently deleted with the replicon. Only the DNA inserted into the multiple cloning sites (passenger genes and homologous fragment for targeting) and a single
FRT
site (68 bp) remain in the chromosome after excision. The utility of these vectors was demonstrated by integrating
Zymomonas mobilis
genes encoding the ethanol pathway behind the native chromosomal
adhE
gene in strains of
E. coli
K-12 and
E. coli
B. With these vectors, a single antibiotic selection system can be used repeatedly for the successive improvement of
E. coli
strains with precise deletion of extraneous genes used during construction.
AlgT (ς
22
) Controls Alginate Production and Tolerance to Environmental Stress in
Pseudomonas syringae
Authors: Keith Lisa M. W. , Bender Carol L. 
Published: December 2019

ABSTRACT

Pseudomonas aeruginosa
and the phytopathogen
P. syringae
produce the exopolysaccharide alginate, which is a copolymer of
d
-mannuronic and
l
-guluronic acids. One of the key regulatory genes controlling alginate biosynthesis in
P. aeruginosa
is
algT
, which encodes the alternate sigma factor, ς
22
. In the present study, the
algT
gene product from
P. syringae
pv. syringae showed 90% amino acid identity with its
P. aeruginosa
counterpart, and sequence analysis of the region flanking
algT
in
P. syringae
revealed the presence of
nadB
,
mucA
, and
mucB
in an arrangement virtually identical to that of
P. aeruginosa
. An
algT
mutant of
P. syringae
was defective in alginate production but could be complemented with wild-type
algT
from
P. syringae
or
P. aeruginosa
when expressed in
trans
. The
algT
mutant also displayed increased sensitivity to heat, paraquat, and hydrogen peroxide (H
2
O
2
); the latter two compounds are known to generate reactive oxygen intermediates. Signals for activation of
algT
gene expression in
P. syringae
were investigated with an
algT
::
uidA
transcriptional fusion. Like that in
P. aeruginosa
,
algT
transcription in
P. syringae
was activated by heat shock. However,
algT
expression in
P. syringae
was also stimulated by osmotic stress and by exposure to paraquat, H
2
O
2
, and copper sulfate. The latter two compounds are frequently encountered during colonization of plant tissue and may be unique signals for
algT
activation in
P. syringae
.
Physical Mapping and Functional Assignment of the Geranylgeranyl-Bacteriochlorophyll Reductase Gene,
bchP
, of
Rhodobacter sphaeroides
Authors: Addlesee Hugh A. , Hunter C. Neil 
Published: December 2019

ABSTRACT

The bacteriochlorophyll of the purple photosynthetic bacterium
Rhodobacter sphaeroides
is esterified with phytol. The presence of this alcohol moiety is essential for the correct assembly of the photosynthetic apparatus. Despite this, and the fact that
R. sphaeroides
is widely used for the study of structure-function relationships in photosynthesis, the molecular genetics of the steps in which the alcohol is added and modified have not previously been investigated in this organism. Sequencing near the center of the photosynthesis gene cluster has now revealed the existence of an open reading frame encoding a putative 394-amino-acid polypeptide displaying strong homology with the products of a number of genes from other photosynthetic organisms, each proposed to be responsible for the reduction of the alcohol moiety of (bacterio)chlorophyll to phytol. An
R. sphaeroides
transposon mutant in this gene,
bchP
, possessed a structurally modified photosystem assembled with bacteriochlorophyll esterified with geranylgeraniol, rather than with phytol, implying that the product of this gene was geranylgeranyl-bacteriochlorophyll reductase. This identification was confirmed by the performance of in vitro assays using heterologously expressed protein, providing the first direct demonstration of the activity of a
bchP
gene product.
Molecular Evidence for a New Bacteriophage of
Borrelia burgdorferi
Authors: Eggers Christian H. , Samuels D. Scott 
Published: December 2019

ABSTRACT

We have recovered a DNase-protected, chloroform-resistant molecule of DNA from the cell-free supernatant of a
Borrelia burgdorferi
culture. The DNA is a 32-kb double-stranded linear molecule that is derived from the 32-kb circular plasmids (cp32s) of the
B. burgdorferi
genome. Electron microscopy of samples from which the 32-kb DNA molecule was purified revealed bacteriophage particles. The bacteriophage has a polyhedral head with a diameter of 55 nm and appears to have a simple 100-nm-long tail. The phage is produced constitutively at low levels from growing cultures of some
B. burgdorferi
strains and is inducible to higher levels with 10 μg of 1-methyl-3-nitroso-nitroguanidine (MNNG) ml
−1
. In addition, the prophage can be induced with MNNG from some
Borrelia
isolates that do not naturally produce phage. We have isolated and partially characterized the phage associated with
B. burgdorferi
CA-11.2A. To our knowledge, this is the first molecular characterization of a bacteriophage of
B. burgdorferi
.