bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cloning and Characterization of the
CSF1
Gene of
Saccharomyces cerevisiae
, Which Is Required for Nutrient Uptake at Low Temperature
Authors: Tokai Masaya , Kawasaki Hideki , Kikuchi Yasuhiro , Ouchi Kozo 
Published: July 2002

ABSTRACT

We have isolated cold-sensitive fermentation mutants (Csf mutants) of a commercial baker's yeast that have practically no fermentation capacity at 5°C and return to their normal capacity at 25 to 40°C.
CSF1
was cloned by functional complementation of the Csf phenotype.
CSF1
contain an open reading frame of 8,874 nucleotides, encoding a protein of 2,958 amino acids. The nucleotide sequence was identical to that of the YLR087C gene in the
Saccharomyces
genome database, but there was no information about the function of the predicted CSF1 (YLR087C) protein. Gene disruption shows that
CSF1
is required for growth and fermentation only at low temperatures. Permeabilized cells of the disruptant showed nearly the same ethanol production rate as those of the parent strain, even at 10°C. The disruptant cells had the same glucose uptake rates as the parental cells at 30°C, but three- to fivefold-lower rates than the parental cells at 10°C. These findings suggest that
CSF1
associates with a new nutrient transport system which exists on the plasma membrane and is required only at low temperature.
Differential Expression of over 60 Chromosomal Genes in
Escherichia coli
by Constitutive Expression of MarA
Authors: Barbosa Teresa M. , Levy Stuart B. 
Published: July 2002

ABSTRACT

In
Escherichia coli
, the MarA protein controls expression of multiple chromosomal genes affecting resistance to antibiotics and other environmental hazards. For a more-complete characterization of the
mar
regulon, duplicate macroarrays containing 4,290 open reading frames of the
E. coli
genome were hybridized to radiolabeled cDNA populations derived from
mar
-deleted and
mar
-expressing
E. coli
. Strains constitutively expressing MarA showed altered expression of more than 60 chromosomal genes: 76% showed increased expression and 24% showed decreased expression. Although some of the genes were already known to be MarA regulated, the majority were newly determined and belonged to a variety of functional groups. Some of the genes identified have been associated with iron transport and metabolism; other genes were previously known to be part of the
soxRS
regulon. Northern blot analysis of selected genes confirmed the results obtained with the macroarrays. The findings reveal that the
mar
locus mediates a global stress response involving one of the largest networks of genes described.
Organization and Cell-Cell Interaction in Starved
Saccharomyces cerevisiae
Colonies
Authors: Varon Mazal , Choder Mordechai 
Published: July 2002

ABSTRACT

Cell growth in yeast colonies is a complex process, the control of which is largely unknown. Here we present scanning electron micrographs of
Saccharomyces cerevisiae
colonies, showing changes in the pattern of cell organization and cell-cell interactions during colony development. In young colonies (≤36 h), cell density is relatively low, and the cells seem to divide in a random orientation. However, as the colonies age, cell density increases and the cells seem to be oriented in a more orderly fashion. Unexpectedly, cells in starved colonies form connecting fibrils. A single connecting fibril 180 ± 50 nm wide is observed between any two neighboring cells, and the fibrils appear to form a global network. The results suggest a novel type of communication between cells within a colony that may contribute to the ability of the community to cope with starvation.
comH
, a Novel Gene Essential for Natural Transformation of
Helicobacter pylori
Authors: Smeets Leonard C. , Bijlsma Jetta J. E. , Boomkens Sacha Y. , Vandenbroucke-Grauls Christina M. J. E. , Kusters Johannes G. 
Published: July 2002

ABSTRACT

Helicobacter pylori
is naturally competent for transformation, but the DNA uptake system of this bacterium is only partially characterized, and nothing is known about the regulation of competence in
H. pylori
. To identify other components involved in transformation or competence regulation in this species, we screened a mutant library for competence-deficient mutants. This resulted in the identification of a novel,
Helicobacter
-specific competence gene (
comH
) whose function is essential for transformation of
H. pylori
with chromosomal DNA fragments as well as with plasmids. Complementation of
comH
mutants in
trans
completely restored competence. Unlike other transformation genes of
H. pylori
,
comH
does not belong to a known family of orthologous genes. Moreover, no significant homologs of
comH
were identified in currently available databases of bacterial genome sequences. The
comH
gene codes for a protein with an N-terminal leader sequence and is present in both highly competent and less-efficient transforming
H. pylori
strains. A
comH
homolog was found in
Helicobacter acinonychis
but not in
Helicobacter felis
and
Helicobacter mustelae.
Slow Polymerization of
Mycobacterium tuberculosis
FtsZ
Authors: White E. Lucile , Ross Larry J. , Reynolds Robert C. , Seitz Lainne E. , Moore Georgia D. , Borhani David W. 
Published: July 2002

ABSTRACT

The essential cell division protein, FtsZ, from
Mycobacterium tuberculosis
has been expressed in
Escherichia coli
and purified. The recombinant protein has GTPase activity typical of tubulin and other FtsZs. FtsZ polymerization was studied using 90° light scattering. The mycobacterial protein reaches maximum polymerization much more slowly (∼10 min) than
E. coli
FtsZ. Depolymerization also occurs slowly, taking 1 h or longer under most conditions. Polymerization requires both Mg
2+
and GTP. The minimum concentration of FtsZ needed for polymerization is 3 μM. Electron microscopy shows that polymerized
M. tuberculosis
FtsZ consists of strands that associate to form ordered aggregates of parallel protofilaments. Ethyl 6-amino-2,3-dihydro-4-phenyl-1H-pyrido[4,3-b][1,4]diazepin-8-ylcarbamate (SRI 7614), an inhibitor of tubulin polymerization synthesized at Southern Research Institute, inhibits
M. tuberculosis
FtsZ polymerization, inhibits GTP hydrolysis, and reduces the number and sizes of FtsZ polymers.
The Serotype of Type Ia and III Group B Streptococci Is Determined by the Polymerase Gene within the Polycistronic Capsule Operon
Authors: Chaffin Donald O. , Beres Stephen B. , Yim Harry H. , Rubens Craig E. 
Published: July 2002

ABSTRACT

Streptococcus agalactiae
is a primary cause of neonatal morbidity and mortality. Essential to the virulence of this pathogen is the production of a type-specific capsular polysaccharide (CPS) that enables the bacteria to evade host immune defenses. The identification, cloning, sequencing, and functional characterization of seven genes involved in type III capsule production have been previously reported. Here, we describe the cloning and sequencing of nine additional adjacent genes,

cps
III
FGHIJKL

,

neu
III
B

, and

neu
III
C

. Sequence comparisons suggested that these genes are involved in sialic acid synthesis, pentasaccharide repeating unit formation, and oligosaccharide transport and polymerization. The type III CPS (
cps
III) locus was comprised of 16 genes within 15.5 kb of contiguous chromosomal DNA. Primer extension analysis and investigation of mRNA from mutants with polar insertions in their
cps
III loci supported the hypothesis that the operon is transcribed as a single polycistronic message. The translated
cps
III sequences were compared to those of the
S. agalactiae cps
Ia locus, and the primary difference between the operons was found to reside in

cps
III
H

, the putative CPS polymerase gene. Expression of

cps
III
H

in a type Ia strain resulted in suppression of CPS Ia synthesis and in production of a CPS which reacted with type III-specific polyclonal antibody. Likewise, expression of the putative type Ia polymerase gene in a type III strain reduced synthesis of type III CPS with production of a type Ia immunoreactive capsule. Based on the similar structures of the oligosaccharide repeating units of the type Ia and III capsules, our observations demonstrated that

cps
Ia
H

and

cps
III
H

encoded the type Ia and III CPS polymerases, respectively. Additionally, these findings suggested that a single gene can confer serotype specificity in organisms that produce complex polysaccharides.
Microarray-Based Identification of a Novel
Streptococcus pneumoniae
Regulon Controlled by an Autoinduced Peptide
Authors: de Saizieu Antoine , Gardès Christophe , Flint Nicholas , Wagner Christian , Kamber Markus , Mitchell Timothy J. , Keck Wolfgang , Amrein Kurt E. , Lange Roland 
Published: July 2002

ABSTRACT

We have identified in the
Streptococcus pneumoniae
genome sequence a two-component system (TCS13, Blp [bacteriocin-like peptide]) which is closely related to quorum-sensing systems regulating cell density-dependent phenotypes such as the development of genetic competence or the production of antimicrobial peptides in lactic acid bacteria. In this study we present evidence that TCS13 is a peptide-sensing system that controls a regulon including genes encoding Blps. Downstream of the Blp TCS (BlpH R) we identified open reading frames (
blpAB
) that have the potential to encode an ABC transporter that is homologous to the ComA/B export system for the competence-stimulating peptide ComC. The putative translation product of
blpC
, a small gene located downstream of
blpAB
, has a leader peptide with a Gly-Gly motif. This leader peptide is typical of precursors processed by this family of transporters. Microarray-based expression profiling showed that a synthetic oligopeptide corresponding to the processed form of BlpC (BlpC*) induces a distinct set of 16 genes. The changes in the expression profile elicited by synthetic BlpC* depend on BlpH since insertional inactivation of its corresponding gene abolishes differential gene induction. Comparison of the promoter regions of the
blp
genes disclosed a conserved sequence element formed by two imperfect direct repeats upstream of extended −10 promoter elements. We propose that BlpH is the sensor for BlpC* and the conserved sequence element is a recognition sequence for the BlpR response regulator.
Domain Structure of
Salmonella
FlhB, a Flagellar Export Component Responsible for Substrate Specificity Switching
Authors: Minamino Tohru , Macnab Robert M. 
Published: July 2002

ABSTRACT

We have investigated the properties of the cytoplasmic domain (FlhB
C
) of the 383-amino-acid
Salmonella
membrane protein FlhB, a component of the type III flagellar export apparatus. FlhB, along with the hook-length control protein FliK, mediates the switching of export specificity from rod- and hook-type substrates to filament-type substrates during flagellar morphogenesis. Wild-type FlhB
C
was unstable (half-life, ca. 5 min), being specifically cleaved at Pro-270 into two polypeptides, FlhB
CN
and FlhB
CC
, which retained the ability to interact with each other after cleavage. Full-length wild-type FlhB was also subject to cleavage. Coproduction of the cleavage products, FlhB
ΔCC
(i.e., the N-terminal transmembrane domain FlhB
TM
plus FlhB
CN
) and FlhB
CC
, resulted in restoration of both motility and flagellar protein export to an
flhB
mutant host, indicating that the two polypeptides were capable of productive association. Mutant FlhB proteins that can undergo switching of substrate specificity even in the absence of FliK were much more resistant to cleavage (half-lives, 20 to 60 min). The cleavage products of wild-type FlhB
C
, existing as a FlhB
CN
–FlhB
CC
complex on an affinity blot membrane, bound the rod- and hook-type substrate FlgD more strongly than the filament-type substrate FliC. In contrast, the intact form of FlhB
C
(mutant or wild type) or the FlhB
CC
polypeptide alone bound FlgD and FliC to about the same extent. FlhB
CN
by itself did not bind substrates appreciably. We propose that FlhB
C
has two substrate specificity states and that a conformational change, mediated by the interaction between FlhB
CN
and FlhB
CC
, is responsible for the specificity switching process. FliK itself is an export substrate; its binding properties for FlhB
C
resemble those of FlgD and do not provide any evidence for a physical interaction beyond that of the export process.
Recognition of Overlapping Nucleotides by AraC and the Sigma Subunit of RNA Polymerase
Authors: Dhiman Anjali , Schleif Robert 
Published: July 2002

ABSTRACT

The
Escherichia coli
promoter

p
BAD

, under the control of the AraC protein, drives the expression of mRNA encoding the AraB, AraA, and AraD gene products of the arabinose operon. The binding site of AraC at

p
BAD

overlaps the RNA polymerase −35 recognition region by 4 bases, leaving 2 bases of the region not contacted by AraC. This overlap raises the question of whether AraC substitutes for the sigma subunit of RNA polymerase in recognition of the −35 region or whether both AraC and sigma make important contacts with the DNA in the −35 region. If sigma does not contact DNA near the −35 region,

p
BAD

activity should be independent of the identity of the bases in the hexamer region that are not contacted by AraC. We have examined this issue in the

p
BAD

promoter and in a second promoter where the AraC binding site overlaps the −35 region by only 2 bases. In both cases promoter activity is sensitive to changes in bases not contacted by AraC, showing that despite the overlap, sigma does read DNA in the −35 region. Since sigma and AraC are thus closely positioned at

p
BAD

, it is possible that AraC and sigma contact one another during transcription initiation. DNA migration retardation assays, however, showed that there exists only a slight degree of DNA binding cooperativity between AraC and sigma, thus suggesting either that the normal interactions between AraC and sigma are weak or that the presence of the entire RNA polymerase is necessary for significant interaction.
Plasmid-Located Pathogenicity Determinants of
Serratia entomophila
, the Causal Agent of Amber Disease of Grass Grub, Show Similarity to the Insecticidal Toxins of
Photorhabdus luminescens
Authors: Hurst Mark R. H. , Glare Travis R. , Jackson Trevor A. , Ronson Clive W. 
Published: July 2002

ABSTRACT

Serratia entomophila
and
Serratia proteamaculans
cause amber disease in the grass grub
Costelytra zealandica
(Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. Larval disease symptoms include cessation of feeding, clearance of the gut, amber coloration, and eventual death. A 115-kb plasmid, pADAP, identified in
S. entomophila
is required for disease causation and, when introduced into
Escherichia coli
, enables that organism to cause amber disease. A 23-kb fragment of pADAP that conferred disease-causing ability on
E. coli
and a pADAP-cured strain of
S. entomophila
was isolated. Using insertion mutagenesis, the pathogenicity determinants were mapped to a 17-kb region of the clone. Sequence analysis of the 17-kb region showed that the predicted products of three of the open reading frames (
sepA
,
sepB
, and
sepC
) showed significant sequence similarity to components of the insecticidal toxin produced by the bacterium
Photorhabdus luminescens
. Transposon insertions in
sepA
,
sepB
, or
sepC
completely abolished both gut clearance and cessation of feeding on the 23-kb clone; when recombined back into pADAP, they abolished gut clearance but not cessation of feeding. These results suggest that SepA, SepB, and SepC together are sufficient for amber disease causation by
S. entomophila
and that another locus also able to exert a cessation-of-feeding effect is encoded elsewhere on pADAP.
The PspA Protein of
Escherichia coli
Is a Negative Regulator of ς
54
-Dependent Transcription
Authors: Dworkin Jonathan , Jovanovic Goran , Model Peter 
Published: July 2002

ABSTRACT

In
Eubacteria
, expression of genes transcribed by an RNA polymerase holoenzyme containing the alternate sigma factor ς
54
is positively regulated by proteins belonging to the family of enhancer-binding proteins (EBPs). These proteins bind to upstream activation sequences and are required for the initiation of transcription at the ς
54
-dependent promoters. They are typically inactive until modified in their N-terminal regulatory domain either by specific phosphorylation or by the binding of a small effector molecule. EBPs lacking this domain, such as the PspF activator of the ς
54
-dependent
pspA
promoter, are constitutively active. We describe here the in vivo and in vitro properties of the PspA protein of
Escherichia coli
, which negatively regulates expression of the
pspA
promoter without binding DNA directly.
Genetics of Swarming Motility inSalmonella entericaSerovar Typhimurium: Critical Role for Lipopolysaccharide
Authors: Toguchi Adam , Siano Michael , Burkart Mark , Harshey Rasika M. 
Published: July 2002

ABSTRACTSalmonella entericaserovar Typhimurium can differentiate into hyperflagellated swarmer cells on agar of an appropriate consistency (0.5 to 0.8%), allowing efficient colonization of the growth surface. Flagella are essential for this form of motility. In order to identify genes involved in swarming, we carried out extensive transposon mutagenesis of serovar Typhimurium, screening for those that had functional flagella yet were unable to swarm. A majority of these mutants were defective in lipopolysaccharide (LPS) synthesis, a large number were defective in chemotaxis, and some had defects in putative two-component signaling components. While the latter two classes were defective in swarmer cell differentiation, representative LPS mutants were not and could be rescued for swarming by external addition of a biosurfactant. A mutation inwaaG(LPS core modification) secreted copious amounts of slime and showed a precocious swarming phenotype. We suggest that the O antigen improves surface “wettability” required for swarm colony expansion, that the LPS core could play a role in slime generation, and that multiple two-component systems cooperate to promote swarmer cell differentiation. The failure to identify specific swarming signals such as amino acids, pH changes, oxygen, iron starvation, increased viscosity, flagellar rotation, or autoinducers leads us to consider a model in which the external slime is itself both the signal and the milieu for swarming motility. The model explains the cell density dependence of the swarming phenomenon.
Multicomponent Transcriptional Regulation at the Complex Promoter of the Exopolysaccharide I Biosynthetic Operon of
Ralstonia solanacearum
Authors: Garg Ram P. , Huang Jianzhong , Yindeeyoungyeon Wandee , Denny Timothy P. , Schell Mark A. 
Published: July 2002

ABSTRACT

High-level transcription of
eps
, an operon encoding biosynthesis of an exopolysaccharide virulence factor of the phytopathogen
Ralstonia
(
Pseudomonas
)
solanacearum
, requires the products of at least seven regulatory genes (
phcA
,
phcB
,
xpsR
,
vsrA-vsrD
, and
vsrB-vsrC
), which are organized in three converging signal transduction cascades. Because
xpsR
and the
vsrB-vsrC
two-component system are the most downstream cascade components required for activation of
eps
, we explored how these components control transcription from the
eps
promoter (P

eps

). Deletion and PCR mutagenesis identified an upstream region of P

eps

(nucleotides −82 to −62) that is critical for transcription activation by VsrB-VsrC and XpsR and also is required for negative control of P

eps

by the putative
eps
regulator EpsR. Using PCR mutagenesis we generated the
vsrC1
allele that encodes a response regulator that constitutively activates P

eps

in the absence of its cognate sensor, VsrB. However, activation of P

eps

by
vsrC1
still required
xpsR
. Unexpectedly, the amino acid substitution conferring the constitutive phenotype on VsrC1 is 12 residues from its C terminus, outside the known functional domains of response regulators. Finally, a modified DNase I footprinting method was used to demonstrate specific binding of both VsrC1 and VsrC to the −72 to −62 upstream region of P

eps

.
Functional Analysis of PvdS, an Iron Starvation Sigma Factor of
Pseudomonas aeruginosa
Authors: Leoni Livia , Orsi Nicola , de Lorenzo Victor , Visca Paolo 
Published: July 2002

ABSTRACT

In
Pseudomonas aeruginosa
, iron modulates gene expression through a cascade of negative and positive regulatory proteins. The master regulator Fur is involved in iron-dependent repression of several genes. One of these genes,
pvdS
, was predicted to encode a putative sigma factor responsible for the transcription of a subset of genes of the Fur regulon. PvdS appears to belong to a structurally and functionally distinct subgroup of the extracytoplasmic function family of alternative sigma factors. Members of this subgroup, also including PbrA from
Pseudomonas fluorescens
, PfrI and PupI from
Pseudomonas putida
, and FecI from
Escherichia coli
, are controlled by the Fur repressor, and they activate transcription of genes for the biosynthesis or the uptake of siderophores. Evidence is provided that the PvdS protein of
P. aeruginosa
is endowed with biochemical properties of eubacterial sigma factors, as it spontaneously forms 1:1 complexes with the core fraction of RNA polymerase (RNAP, α
2
ββ′ subunits), thereby promoting in vitro binding of the PvdS-RNAP holoenzyme to the promoter region of the
pvdA
gene. These functional features of PvdS are consistent with the presence of structural domains predicted to be involved in core RNAP binding, promoter recognition, and open complex formation. The activity of pyoverdin biosynthetic (
pvd
) promoters was significantly lower in
E. coli
overexpressing the multicopy
pvdS
gene than in wild-type
P. aeruginosa
PAO1 carrying the single gene copy, and
pvd
::
lacZ
transcriptional fusions were silent in both
pfrI
(the
pvdS
homologue) and
pfrA
(a positive regulator of pseudobactin biosynthetic genes) mutants of
P. putida
WCS358, while they are expressed at PAO1 levels in wild-type WCS358. Moreover, the PvdS-RNAP holoenzyme purified from
E. coli
lacked the ability to generate in vitro transcripts from the
pvdA
promoter. These observations suggest that at least one additional positive regulator could be required for full activity of the PvdS-dependent transcription complex both in vivo and in vitro. This is consistent with the presence of a putative activator binding site (the iron starvation box) at variable distance from the transcription initiation sites of promoters controlled by the iron starvation sigma factors PvdS, PfrI, and PbrA of fluorescent pseudomonads.
CelE, a Multidomain Cellulase from
Clostridium cellulolyticum
: a Key Enzyme in the Cellulosome?
Authors: Gaudin Christian , Belaich Anne , Champ Stéphanie , Belaich Jean-Pierre 
Published: July 2002

ABSTRACT

CelE, one of the three major proteins of the cellulosome of
Clostridium cellulolyticum
, was characterized. The amino acid sequence of the protein deduced from
celE
DNA sequence led us to the supposition that CelE is a three-domain protein. Recombinant CelE and a truncated form deleted of the putative cellulose binding domain (CBD) were obtained. Deletion of the CBD induces a total loss of activity. Exhibiting rather low levels of activity on soluble, amorphous, and crystalline celluloses, CelE is more active on
p
-nitrophenyl–cellobiose than the other cellulases from this organism characterized to date. The main product of its action on Avicel is cellobiose (more than 90% of the soluble sugars released), and its attack on carboxymethyl cellulose is accompanied by a relatively small decrease in viscosity. All of these features suggest that CelE is a cellobiohydrolase which has retained a certain capacity for random attack mode. We measured saccharification of Avicel and bacterial microcrystalline cellulose by associations of CelE with four other cellulases from
C. cellulolyticum
and found that CelE acts synergistically with all tested enzymes. The positive influence of CelE activity on the activities of other cellulosomal enzymes may explain its relative abundance in the cellulosome.