bluuebunny/crossref_metadata_2025_split huggingface dataset:
Specificity of Signal Peptide Recognition in Tat-Dependent Bacterial Protein Translocation
Authors: Blaudeck Natascha , Sprenger Georg A. , Freudl Roland , Wiegert Thomas 
Published: July 2002

ABSTRACT

The bacterial twin arginine translocation (Tat) pathway translocates across the cytoplasmic membrane folded proteins which, in most cases, contain a tightly bound cofactor. Specific amino-terminal signal peptides that exhibit a conserved amino acid consensus motif, S/T-R-R-X-F-L-K, direct these proteins to the Tat translocon. The glucose-fructose oxidoreductase (GFOR) of
Zymomonas mobilis
is a periplasmic enzyme with tightly bound NADP as a cofactor. It is synthesized as a cytoplasmic precursor with an amino-terminal signal peptide that shows all of the characteristics of a typical twin arginine signal peptide. However, GFOR is not exported to the periplasm when expressed in the heterologous host
Escherichia coli
, and enzymatically active pre-GFOR is found in the cytoplasm. A precise replacement of the pre-GFOR signal peptide by an authentic
E. coli
Tat signal peptide, which is derived from pre-trimethylamine
N
-oxide (TMAO) reductase (TorA), allowed export of GFOR, together with its bound cofactor, to the
E. coli
periplasm. This export was inhibited by carbonyl cyanide
m
-chlorophenylhydrazone, but not by sodium azide, and was blocked in
E. coli tatC
and
tatAE
mutant strains, showing that membrane translocation of the TorA-GFOR fusion protein occurred via the Tat pathway and not via the Sec pathway. Furthermore, tight cofactor binding (and therefore correct folding) was found to be a prerequisite for proper translocation of the fusion protein. These results strongly suggest that Tat signal peptides are not universally recognized by different Tat translocases, implying that the signal peptides of Tat-dependent precursor proteins are optimally adapted only to their cognate export apparatus. Such a situation is in marked contrast to the situation that is known to exist for Sec-dependent protein translocation.
Expression of the
Bacillus subtilis trpEDCFBA
Operon Is Influenced by Translational Coupling and Rho Termination Factor
Authors: Yakhnin Helen , Babiarz Joshua E. , Yakhnin Alexander V. , Babitzke Paul 
Published: July 2002

ABSTRACT

The
trp
RNA-binding attenuation protein (TRAP) regulates expression of the
Bacillus subtilis trpEDCFBA
operon by transcription attenuation and translational control mechanisms. Both mechanisms require binding of tryptophan-activated TRAP to 11 (G/U)AG repeats in the
trp
leader transcript.
trpE
translational control involves formation of a TRAP-dependent RNA structure that sequesters the
trpE
Shine-Dalgarno (SD) sequence (the SD blocking hairpin). By comparing expression levels from
trpE
′
-
′
lacZ
translational fusions controlled by the wild-type leader or by a leader that cannot form the SD blocking hairpin, we found that translational control requires a tryptophan concentration higher than that required for transcription attenuation. We also found that inhibition of
trpE
translation by the SD blocking hairpin does not alter the stability of the downstream message. Since the coding sequences for
trpE
and
trpD
overlap by 29 nucleotides, we examined expression levels from
trpED
′
-
′
lacZ
translational fusions to determine if these two genes are translationally coupled. We found that introduction of a UAA stop codon in
trpE
resulted in a substantial reduction in expression. Since expression was partially restored in the presence of a tRNA suppressor, our results indicate that
trpE
and
trpD
are translationally coupled. We determined that the coupling mechanism is TRAP independent and that formation of the SD blocking hairpin regulates
trpD
translation via translational coupling. We also constructed a
rho
mutation to investigate the role of Rho-dependent termination in
trp
operon expression. We found that TRAP-dependent formation of the SD blocking hairpin allows Rho access to the nascent transcript, causing transcriptional polarity.
Bacterial Two-Hybrid Analysis of Interactions between Region 4 of the ς
70
Subunit of RNA Polymerase and the Transcriptional Regulators Rsd from
Escherichia coli
and AlgQ from
Pseudomonas aeruginosa
Authors: Dove Simon L. , Hochschild Ann 
Published: July 2002

ABSTRACT

A number of transcriptional regulators mediate their effects through direct contact with the ς
70
subunit of
Escherichia coli
RNA polymerase (RNAP). In particular, several regulators have been shown to contact a C-terminal portion of ς
70
that harbors conserved region 4. This region of ς contains a putative helix-turn-helix DNA-binding motif that contacts the −35 element of ς
70
-dependent promoters directly. Here we report the use of a recently developed bacterial two-hybrid system to study the interaction between the putative anti-ς factor Rsd and the ς
70
subunit of
E. coli
RNAP. Using this system, we found that Rsd can interact with an 86-amino-acid C-terminal fragment of ς
70
and also that amino acid substitution R596H, within region 4 of ς
70
, weakens this interaction. We demonstrated the specificity of this effect by showing that substitution R596H does not weaken the interaction between ς and two other regulators shown previously to contact region 4 of ς
70
. We also demonstrated that AlgQ, a homolog of Rsd that positively regulates virulence gene expression in
Pseudomonas aeruginosa,
can contact the C-terminal region of the ς
70
subunit of RNAP from this organism. We found that amino acid substitution R600H in ς
70
from
P. aeruginosa
, corresponding to the R596H substitution in
E. coli
ς
70
, specifically weakens the interaction between AlgQ and ς
70
. Taken together, our findings suggest that Rsd and AlgQ contact similar surfaces of RNAP present in region 4 of ς
70
and probably regulate gene expression through this contact.
Interaction of MutS and Vsr: Some Dominant-Negative
mutS
Mutations That Disable Methyladenine-Directed Mismatch Repair Are Active in Very-Short-Patch Repair
Authors: Lieb Margaret , Rehmat Shehnaz , Bhagwat Ashok S. 
Published: July 2002

ABSTRACT

In
Escherichia coli
and related bacteria, the very-short-patch (VSP) repair pathway uses an endonuclease, Vsr, to correct T · G mismatches that result from the deamination of 5-methylcytosines in DNA to C · G. The products of
mutS
and
mutL
, which are required for adenine methylation-directed mismatch repair (MMR), enhance VSP repair. Multicopy plasmids carrying
mutS
alleles that are dominant negative for MMR were tested for their effects on VSP repair. Some
mutS
mutations (class I) did not lower VSP repair in a
mutS
+
background, and most class I mutations increased VSP repair in
mutS
cells more than plasmids containing
mutS
+
. Other plasmid-borne
mutS
mutations (class II) and
mutS
+
decreased VSP repair in the
mutS
+
background. Thus, MutS protein lacking functions required for MMR can still participate in VSP repair, and our results are consistent with a model in which MutS binds transiently to the mispair and then translocates away from the mispair to create a specialized structure that enhances the binding of Vsr.
A Eukaryotic-Type Protein Kinase, SpkA, Is Required for Normal Motility of the Unicellular Cyanobacterium
Synechocystis
sp. Strain PCC 6803
Authors: Kamei Ayako , Yuasa Takashi , Orikawa Kumi , Geng Xiao Xing , Ikeuchi Masahiko 
Published: July 2002

ABSTRACT

The genome of the unicellular cyanobacterium
Synechocystis
sp. strain PCC 6803 comprises many open reading frames (ORFs) which putatively encode eukaryotic-type protein kinase and protein phosphatase. Based on gene disruption analysis, a region of the hypothetical ORF
sll1575
, which retained a part of the protein kinase motif, was found to be required for normal motility in the original isolate of strain PCC 6803. Sequence determination revealed that in this strain
sll1575
was part of a gene (designated
spkA
) which harbored an entire eukaryotic-type Ser/Thr protein kinase motif. Strain ATCC 27184 and a glucose-tolerant strain derived from the same isolate as the PCC strain had a frameshift mutation dividing
spkA
into ORFs
sll1574
and
sll1575
. The structural integrity of
spkA
agreed well with the motility phenotype, determined by colony morphology on agar plates. The
spkA
gene was expressed in
Escherichia coli
as a His-tagged protein, which was purified by Ni
2+
affinity chromatography. With [γ-
32
P]ATP, SpkA was autophosphorylated and transferred the phosphate group to casein, myelin basic protein, and histone. SpkA also phosphorylated several proteins in the membrane fraction of
Synechocystis
cells. These results suggest that SpkA is a eukaryotic-type Ser/Thr protein kinase and regulates cellular motility via phosphorylation of the membrane proteins in
Synechocystis
.
Bacillus subtilis
NhaC, an Na
+
/H
+
Antiporter, Influences Expression of the
phoPR
Operon and Production of Alkaline Phosphatases
Authors: Prágai Zoltán , Eschevins Caroline , Bron Sierd , Harwood Colin R. 
Published: July 2002

ABSTRACT

When
Bacillus subtilis
is subjected to phosphate starvation, genes of the Pho regulon are either induced or repressed. Among those induced are genes encoding alkaline phosphatases (APases). A set of isogenic mutants, with a β-galactosidase gene transcriptionally fused to the inactivated target gene, was used to identify genes that influence the operation of the Pho regulon. One such gene was
nhaC
(previously
yheL
). In the absence of NhaC, growth and APase production were enhanced, while the production of other non-Pho-regulon secretory proteins (proteases and α-amylase) did not change. The influence of NhaC on growth, APase synthesis, and its own expression was dependent on the external Na
+
concentration. Other monovalent cations such as Li
+
or K
+
had no effect. We propose a role for NhaC in the uptake of Na
+
.
nhaC
appears to be encoded by a monocistronic operon and, contrary to previous reports, is not in the same transcriptional unit as
yheK
, the gene immediately upstream. The increase in APase production was dependent on an active PhoR, the sensor kinase of the two-component system primarily responsible for controlling the Pho regulon. Transcriptional fusions showed that the
phoPR
operon and both
phoA
(encoding APaseA) and
phoB
(encoding APaseB) were hyperinduced in the absence of NhaC and repressed when this protein was overproduced. This suggests that NhaC effects APase production via
phoPR
.
Mutational Scanning and Affinity Cleavage Analysis of UhpA-Binding Sites in the
Escherichia coli uhpT
Promoter
Authors: Olekhnovich Igor N. , Kadner Robert J. 
Published: July 2002

ABSTRACT

UhpA, a member of the NarL family of response regulators, activates transcription of the
Escherichia coli uhpT
gene for the sugar phosphate transporter UhpT in response to extracellular glucose-6-phosphate. UhpA binds with different affinities to adjacent regions in the
uhpT
promoter, termed the strong-binding (S) region from −80 to −50 and the weak-binding (W) region from −50 to −32. Transcription activation by UhpA is stimulated by the catabolite gene activator protein (CAP)-cyclic AMP complex and depends on the C-terminal domains of the RNA polymerase RpoA and RpoD subunits. Because single-base substitutions in the UhpA-binding region had little effect on promoter activity, nucleotide substitutions in successive 4-bp blocks throughout this region were examined for their effects on promoter activation and UhpA binding. Changes in three of four blocks within the W region substantially impaired the ability of UhpA to bind to this region, to drive expression of a
uhpT-lacZ
reporter, and to support UhpA-dependent in vitro transcription. These W region variant promoters were strongly stimulated by CAP. Changes in several parts of the S region impaired UhpA binding to both the S and W regions and decreased promoter activity in vivo and in vitro. Thus, binding of UhpA to the W region is crucial for UhpA-dependent activation and depends on occupancy of the S region. None of these substitutions eliminated promoter function. The orientation of UhpA-binding sites was assessed by the affinity cleavage method. The iron chelate FeBABE [iron (
S
)-1-(
p
-bromoacetamidobenzyl) EDTA] was covalently attached to engineered cysteine residues near the DNA-binding region in UhpA. Hydroxyl radicals generated by the iron chelate attached at position 187 resulted in DNA strand cleavages in two clusters of sites located in the middle of the S and W regions. These results are consistent with the binding of two dimers of UhpA. Each dimer binds to an inverted repeat of monomer-binding sites with the consensus sequence CCTGRR, where R is A or G, and each is separated by 6 bp. It is likely that members of the NarL family bind to dyad targets, in contrast to the binding of OmpR family response regulators to direct-repeat targets.
Activation of Prophage
eib
Genes for Immunoglobulin-Binding Proteins by Genes from the IbrAB Genetic Island of
Escherichia coli
ECOR-9
Authors: Sandt Carol H. , Hopper James E. , Hill Charles W. 
Published: July 2002

ABSTRACT

Four distinct
Escherichia coli
immunoglobulin-binding (
eib
) genes, each of which encodes a surface-exposed protein that binds immunoglobulins in a nonimmune manner, are carried by separate prophages in
E. coli
reference (ECOR) strain ECOR-9. Each
eib
gene was transferred to test
E. coli
strains, both in the form of multicopy recombinant plasmids and as lysogenized prophage. The derived lysogens express little or no Eib protein, in sharp contrast to the parental lysogen, suggesting that ECOR-9 has an expression-enhancing activity that the derived lysogens lack. Supporting this hypothesis, we cloned from ECOR-9 overlapping genes,
ibrA
and
ibrB
(designation is derived from “immunoglobulin-binding regulator”), which together activated
eib
expression in the derived lysogens. The proteins encoded by
ibrA
and
ibrB
are very similar to uncharacterized proteins encoded by genes of
Salmonella enterica
serovar Typhi and
E. coli
O157:H7 (in a prophage-like element of the Sakai strain and in two O islands of strain EDL933). The genomic segment containing
ibrA
and
ibrB
has been designated the IbrAB island. It contains regions of homology to the Shiga toxin-converting prophage, Stx2, as well as genes homologous to phage antirepressor genes. The left boundary between the IbrAB island and the chromosomal framework is located near min 35.8 of the
E. coli
K-12 genome. Homology to IbrAB was found in certain other ECOR strains, including the other five
eib
-positive strains and most strains of the phylogenetic group B2. Sequencing of a 1.1-kb portion of
ibrAB
revealed that the other
eib
-positive strains diverge by ≤0.1% from ECOR-9, whereas
eib
-negative ECOR-47 diverges by 16%.
Gene Transfer in
Mycoplasma pulmonis
Authors: Teachman Amy M. , French C. Todd , Yu Huilan , Simmons Warren L. , Dybvig Kevin 
Published: July 2002

ABSTRACT

Experiments were undertaken to examine gene transfer in
Mycoplasma pulmonis
. Parent strains containing transposon-based tetracycline and chloramphenicol resistance markers were combined to allow transfer of markers. Two mating protocols were developed. The first consisted of coincubating the strains in broth culture for extended periods of time. The second protocol consisted of a brief incubation of the combined strains in a 50% solution of polyethylene glycol. Using either protocol, progeny that had acquired antibiotic resistance markers from both parents were obtained. Analysis of the progeny indicated that only the transposon and not flanking genomic DNA was transferred to the recipient cell. Gene transfer was DNase resistant and probably the result of conjugation or cell fusion.
Analyses of the Roles of the Three
cheA
Homologs in Chemotaxis of
Vibrio cholerae
Authors: Gosink Khoosheh K. , Kobayashi Reiji , Kawagishi Ikuro , Häse Claudia C. 
Published: July 2002

ABSTRACT

The
Vibrio cholerae
genome revealed the presence of multiple sets of chemotaxis genes, including three
cheA
gene homologs. We found that the
cheA
-2, but not
cheA
-1 or
cheA
-3, gene is essential for chemotaxis under standard conditions. Loss of chemotaxis had no effect on virulence factor expression in vitro.
Complete Genomic Sequence of SfV, a Serotype-Converting Temperate Bacteriophage of
Shigella
flexneri
Authors: Allison Gwen E. , Angeles Dario , Tran-Dinh Nai , Verma Naresh K. 
Published: July 2002

ABSTRACT

Bacteriophage SfV is a temperate serotype-converting phage of
Shigella
flexneri.
SfV encodes the factors involved in type V O-antigen modification, and the serotype conversion and integration-excision modules of the phage have been isolated and characterized. We now report on the complete sequence of the SfV genome (37,074 bp). A total of 53 open reading frames were predicted from the nucleotide sequence, and analysis of the corresponding proteins was used to construct a functional map. The general organization of the genes in the SfV genome is similar to that of bacteriophage λ, and numerous features of the sequence are described. The superinfection immunity system of SfV includes a lambda-like repression system and a P4-like transcription termination mechanism. Sequence analysis also suggests that SfV encodes multiple DNA methylases, and experiments confirmed that
orf-41
encodes a Dam methylase. Studies conducted to determine if the phage-encoded methylase confers host DNA methylation showed that the two
S.
flexneri
strains analyzed encode their own Dam methylase. Restriction mapping and sequence analysis revealed that the phage genome has
cos
sites at the termini. The tail assembly and structural genes of SfV show homology to those of phage Mu and Mu-like prophages in the genome of
Escherichia
coli
O157:H7 and
Haemophilus
influenzae
. Significant homology (30% of the genome in total) between sections of the early, regulatory, and structural regions of the SfV genome and the e14 and KpLE1 prophages in the
E.
coli
K-12 genome were noted, suggesting that these three phages have common evolutionary origins.
Novel Carbohydrate-Binding Module of β-1,3-Xylanase from a Marine Bacterium,
Alcaligenes
sp. Strain XY-234
Authors: Okazaki Fumiyoshi , Tamaru Yutaka , Hashikawa Shinnosuke , Li Yu-Teh , Araki Toshiyoshi 
Published: July 2002

ABSTRACT

A β-1,3-xylanase gene (
txyA
) from a marine bacterium,
Alcaligenes
sp. strain XY-234, has been cloned and sequenced.
txyA
consists of a 1,410-bp open reading frame that encodes 469 amino acid residues with a calculated molecular mass of 52,256 Da. The domain structure of the β-1,3-xylanase (TxyA) consists of a signal peptide of 22 amino acid residues, followed by a catalytic domain which belongs to family 26 of the glycosyl hydrolases, a linker region with one array of DGG and six repeats of DNGG, and a novel carbohydrate-binding module (CBM) at the C terminus. The recombinant TxyA hydrolyzed β-1,3-xylan but not other polysaccharides such as β-1,4-xylan, carboxymethylcellulose, curdlan, glucomannan, or β-1,4-mannan. TxyA was capable of binding specifically to β-1,3-xylan. The analysis using truncated TxyA lacking either the N- or C-terminal region indicated that the region encoding the CBM was located between residues 376 and 469. Binding studies on the CBM revealed that the

K
d

and the maximum amount of protein bound to β-1,3-xylan were 4.2 μM and 18.2 μmol/g of β-1,3-xylan, respectively. Furthermore, comparison of the enzymatic properties between proteins with and without the CBM strongly indicated that the CBM of TxyA plays an important role in the hydrolysis of β-1,3-xylan.
Cloning of the
sodA
Gene from
Corynebacterium melassecola
and Role of Superoxide Dismutase in Cellular Viability
Authors: Merkamm Muriel , Guyonvarch Armel 
Published: July 2002

ABSTRACT

The
sodA
gene encoding the
Corynebacterium melassecola
manganese-cofactored superoxide dismutase (SOD) has been cloned in
Escherichia coli
and sequenced. The gene is transcribed monocistronically; the predicted polypeptide is 200 amino acids long and associates in a homotetrameric, manganese-dependent form, able to complement an SOD-deficient
E. coli
mutant. A second open reading frame, coding for a putative 217-amino-acid protein with high homology to peptide methionine sulfoxide reductases from various origins, has been identified immediately upstream of
sodA
in the opposite transcription orientation. The
sodA
gene was inactivated by insertion of an integrative vector carrying a kanamycin resistance gene. The growth rate of the SOD-deficient integrant was only slightly affected in BHI rich medium as well as in BMCG chemically defined medium, but was strongly affected by the presence of the redox-cycling agent paraquat. The SOD deficiency had, on the other hand, a deleterious effect on viability as soon as the culture entered the stationary phase of growth in BHI medium. Surprisingly, SOD deficiency was able to rescue the dramatic loss of viability observed for the wild-type strain in BMCG synthetic medium when glucose was not the limiting growth factor.
Molecular Typing of
Salmonella
Serotypes Prevalent in Animals in England: Assessment of Methodology
Authors: Liebana Ernesto , Guns Daniel , Garcia-Migura Lourdes , Woodward Martin J. , Clifton-Hadley Felicity A. , Davies Robert H. 
Published: July 2002

ABSTRACT

Salmonella
enterica
serotypes Derby, Mbandaka, Montevideo, Livingstone, and Senftenberg were among the 10 most prevalent serotypes isolated from farm animals in England and Wales in 1999. These serotypes are of potential zoonotic relevance; however, there is currently no “gold standard” fingerprinting method for them. A collection of isolates representing the former serotypes and serotype Gold Coast were analyzed using plasmid profiling, pulsed-field gel electrophoresis (PFGE), and ribotyping. The success of the molecular methods in identifying DNA polymorphisms was different for each serotype. Plasmid profiling was particularly useful for serotype Derby isolates, and it also provided a good level of discrimination for serotype Senftenberg. For most serotypes, we observed a number of nontypeable plasmid-free strains, which represents a limitation of this technique. Fingerprinting of genomic DNA by ribotyping and PFGE produced a significant variation in results, depending on the serotype of the strain. Both
Pst
I/
Sph
I ribotyping and
Xba
I-PFGE provided a similar degree of strain differentiation for serotype Derby and serotype Senftenberg, only marginally lower than that achieved by plasmid profiling. Ribotyping was less sensitive than PFGE when applied to serotype Mbandaka or serotype Montevideo. Serotype Gold Coast isolates were found to be nontypeable by
Xba
I-PFGE, and a significant proportion of them were found to be plasmid free. A similar situation applies to a number of serotype Livingstone isolates which were nontypeable by plasmid profiling and/or PFGE. In summary, the serotype of the isolates has a considerable influence in deciding the best typing strategy; a single method cannot be relied upon for discriminating between strains, and a combination of typing methods allows further discrimination.