bluuebunny/crossref_metadata_2025_split huggingface dataset:
Polar Clustering of the Chemoreceptor Complex in
Escherichia coli
Occurs in the Absence of Complete CheA Function
Authors: Skidmore J. M. , Ellefson D. D. , McNamara B. P. , Couto M. M. P. , Wolfe A. J. , Maddock J. R. 
Published: July 2002

ABSTRACT

Bacterial chemotaxis requires a phosphorelay system initiated by the interaction of a ligand with its chemoreceptor and culminating in a change in the directional bias of flagellar rotation. Chemoreceptor-CheA-CheW ternary complexes mediate transduction of the chemotactic signal. In vivo, these complexes cluster predominantly in large groups at the cell poles. The function of chemoreceptor clustering is currently unknown. To gain insight into the relationship between signaling and chemoreceptor clustering, we examined these properties in several
Escherichia coli
mutant strains that produce CheA variants altered in their ability to mediate chemotaxis, autophosphorylate, or bind ATP. We show here that polar clustering of chemoreceptor complexes does not require functional CheA protein, although maximal clustering occurred only in chemotactically competent cells. Surprisingly, in cells containing a minimum of 13 gold particles at the cell pole, a significant level of clustering was observed in the absence of CheA, demonstrating that CheA is not absolutely essential for chemoreceptor clustering. Nonchemotactic cells expressing only CheA
S
, a C-terminal CheA deletion, or CheA bearing a mutation in the ATP-binding site mediated slightly less than maximal chemoreceptor clustering. Cells expressing only full-length CheA (CheA
L
) from either a chromosomal or a plasmid-encoded allele displayed a methyl-accepting chemotaxis protein localization pattern indistinguishable from that of strains carrying both CheA
L
and CheA
S
, demonstrating that CheA
L
alone can mediate polar clustering.
Role of Cell Shape in Determination of the Division Plane in
Schizosaccharomyces pombe
: Random Orientation of Septa in Spherical Cells
Authors: Sipiczki M. , Yamaguchi M. , Grallert A. , Takeo K. , Zilahi E. , Bozsik A. , Miklos I. 
Published: July 2002

ABSTRACT

The establishment of growth polarity in
Schizosaccharomyces pombe
cells is a combined function of the cytoplasmic cytoskeleton and the shape of the cell wall inherited from the mother cell. The septum that divides the cylindrical cell into two siblings is formed midway between the growing poles and perpendicularly to the axis that connects them. Since the daughter cells also extend at their ends and form their septa at right angles to the longitudinal axis, their septal (division) planes lie parallel to those of the mother cell. To gain a better understanding of how this regularity is ensured, we investigated septation in spherical cells that do not inherit morphologically predetermined cell ends to establish poles for growth. We studied four mutants (defining four novel genes), over 95% of whose cells displayed a completely spherical morphology and a deficiency in mating and showed a random distribution of cytoplasmic microtubules, Tea1p, and F-actin, indicating that the cytoplasmic cytoskeleton was poorly polarized or apolar. Septum positioning was examined by visualizing septa and division scars by calcofluor staining and by the analysis of electron microscopic images. Freeze-substitution, freeze-etching, and scanning electron microscopy were used. We found that the elongated bipolar shape is not essential for the determination of a division plane that can separate the postmitotic nuclei. However, it seems to be necessary for the maintenance of the parallel orientation of septa over the generations. In the spherical cells, the division scars and septa usually lie at angles to each other on the cell surface. We hypothesize that the shape of the cell indirectly affects the positioning of the septum by directing the extension of the spindle.
Cellular Responses to Postsegregational Killing by Restriction-Modification Genes
Authors: Handa Naofumi , Ichige Asao , Kusano Kohji , Kobayashi Ichizo 
Published: July 2002

ABSTRACT

Plasmids that carry one of several type II restriction modification gene complexes are known to show increased stability. The underlying mechanism was proposed to be the lethal attack by restriction enzyme at chromosomal recognition sites in cells that had lost the restriction modification gene complex. In order to examine bacterial responses to this postsegregational cell killing, we analyzed the cellular processes following loss of the
Eco
RI restriction modification gene complex carried by a temperature-sensitive plasmid in an
Escherichia coli
strain that is wild type with respect to DNA repair. A shift to the nonpermissive temperature blocked plasmid replication, reduced the increase in viable cell counts and resulted in loss of cell viability. Many cells formed long filaments, some of which were multinucleated and others anucleated. In a mutant defective in RecBCD exonuclease/recombinase, these cell death symptoms were more severe and cleaved chromosomes accumulated. Growth inhibition was also more severe in
recA
,
ruvAB
,
ruvC
,
recG
, and
recN
mutants. The cells induced the SOS response in a RecBC-dependent manner. These observations strongly suggest that bacterial cells die as a result of chromosome cleavage after loss of a restriction modification gene complex and that the bacterial RecBCD/RecA machinery helps the cells to survive, at least to some extent, by repairing the cleaved chromosomes. These and previous results have led us to hypothesize that the RecBCD/Chi/RecA system serves to destroy restricted “nonself” DNA and repair restricted “self” DNA.
Cloning and Sequence Analysis of the Mercury Resistance Operon of
Streptomyces
sp. Strain CHR28 Reveals a Novel Putative Second Regulatory Gene
Authors: Ravel Jacques , DiRuggiero Jocelyne , Robb Frank T. , Hill Russell T. 
Published: July 2002

ABSTRACT

A DNA library of pRJ28, a large linear plasmid encoding mercury resistance, was constructed, and the mercury resistance genes were cloned. The 5,921-bp sequence was analyzed and showed a high degree of similarity to the
Streptomyces lividans
1326 mercury resistance operon. Genes
merR
,
merT
,
merP
, and
orfIV
were found in a similar order and in a single transcription unit.
merA
and
merB
were found to be transcribed in the opposite direction to genes
merR
,
merT
,
merP
, and
orfIV
, as in
S. lividans
1326. A novel putative regulatory gene,
orfX
, was found 22 bp downstream of
merA. orfX
encodes a 137-amino acid protein with a potential helix-turn-helix motif in the N-terminal domain, characteristic of the MerR family of transcriptional regulators. Transcriptional studies showed that
orfX
is cotranscribed with
merA
and
merB
. It is hypothesized that
orfX
plays a role in the regulation of the mercury resistance operon, probably by binding at the MerR operator site.
Equivalence of Lauric Acid and Glycerol Monolaurate as Inhibitors of Signal Transduction in
Staphylococcus aureus
Authors: Ruzin Alexey , Novick Richard P. 
Published: July 2002

ABSTRACT

Glycerol monolaurate (GML) inhibits the expression of virulence factors in
Staphylococus aureus
and the induction of vancomycin resistance in
Enterococcus faecalis
, presumably by blocking signal transduction. Although GML is rapidly hydrolyzed by bacteria, one of the products, lauric acid, has identical inhibitory activity and is metabolized much more slowly. At least four distinct GML-hydrolyzing activities are identified in
S. aureus
: the secreted Geh lipase, residual supernatant activity in a
geh
-null mutant strain, a novel membrane-bound esterase, and a cytoplasmic activity.
Promoter Cloning in the Radioresistant Bacterium
Deinococcus radiodurans
Authors: Meima Rob , Rothfuss Heather M. , Gewin Lindy , Lidstrom Mary E. 
Published: July 2002

ABSTRACT

Deinococcus radiodurans
is a highly radiation-resistant bacterium that is classed in a major subbranch of the bacterial domain. Since very little is known about gene expression in this bacterium, an initial study of promoters was undertaken. In order to isolate promoters and study promoter function, a series of integrative vectors for stable chromosomal insertion in
D. radiodurans
were developed. These vectors are based on
Escherichia coli
replicons that are unable to replicate autonomously in
D. radiodurans
and carry homologous sequences for replacement recombination in the
D. radiodurans
chromosome. The resulting integration vectors were used to study expression of reporter genes fused to a number of putative promoters that were amplified from the
D. radiodurans
R1 genome. Further analysis of these and other putative promoters was performed by Northern hybridization and primer extension experiments. In contrast to previous reports, the −10 and −35 regions of these promoters resembled the ς
70
consensus sequence of
E. coli
.
Role of Acid Metabolism in
Streptomyces coelicolor
Morphological Differentiation and Antibiotic Biosynthesis
Authors: Viollier Patrick H. , Minas Wolfgang , Dale Glenn E. , Folcher Marc , Thompson Charles J. 
Published: July 2002

ABSTRACT

Studies of citrate synthase (CitA) were carried out to investigate its role in morphological development and biosynthesis of antibiotics in
Streptomyces coelicolor
. Purification of CitA, the major vegetative enzyme activity, allowed characterization of its kinetic properties. The apparent
K

m

values of CitA for acetyl coenzyme A (acetyl-CoA) (32 μM) and oxaloacetate (17 μM) were similar to those of citrate synthases from other gram-positive bacteria and eukaryotes. CitA was not strongly inhibited by various allosteric feedback inhibitors (NAD
+
, NADH, ATP, ADP, isocitrate, or α-ketoglutarate). The corresponding gene (
citA
) was cloned and sequenced, allowing construction of a
citA
mutant (BZ2). BZ2 was a glutamate auxotroph, indicating that
citA
encoded the major citrate synthase allowing flow of acetyl-CoA into the tricarboxylic acid (TCA) cycle. Interruption of aerobic TCA cycle-based metabolism resulted in acidification of the medium and defects in morphological differentiation and antibiotic biosynthesis. These developmental defects of the
citA
mutant were in part due to a glucose-dependent medium acidification that was also exhibited by some other bald mutants. Unlike other acidogenic bald strains,
citA
and
bldJ
mutants were able to produce aerial mycelia and pigments when the medium was buffered sufficiently to maintain neutrality. Extracellular complementation studies suggested that
citA
defines a new stage of the
Streptomyces
developmental cascade.
Specificity and Topology of the
Escherichia coli
Xanthosine Permease, a Representative of the NHS Subfamily of the Major Facilitator Superfamily
Authors: Nørholm Morten H. H. , Dandanell Gert 
Published: July 2002

ABSTRACT

The specificity of XapB permease was compared with that of the known nucleoside transporters NupG and NupC. XapB-mediated xanthosine uptake is abolished by 2,4-dinitrophenol and exhibits saturation kinetics with an apparent

K
m

of 136 μM. A 12-transmembrane-segment model was confirmed by translational fusions to alkaline phosphatase and the α fragment of β-galactosidase.
High-Density Microarray-Mediated Gene Expression Profiling of
Escherichia coli
Authors: Wei Yan , Lee Jian-Ming , Richmond Craig , Blattner Frederick R. , Rafalski J. Antoni , LaRossa Robert A. 
Published: July 2002

ABSTRACT

A nearly complete collection of 4,290
Escherichia coli
open reading frames was amplified and arrayed in high density on glass slides. To exploit this reagent, conditions for RNA isolation from
E. coli
cells, cDNA production with attendant fluorescent dye incorporation, DNA-DNA hybridization, and hybrid quantitation have been established. A brief isopropyl-β-
d
-thiogalactopyranoside (IPTG) treatment elevated
lacZ
,
lacY
, and
lacA
transcript content about 30-fold; in contrast, most other transcript titers remained unchanged. Distinct RNA expression patterns between
E. coli
cultures in the exponential and transitional phases of growth were catalogued, as were differences associated with culturing in minimal and rich media. The relative abundance of each transcript was estimated by using hybridization of a genomic DNA-derived, fluorescently labeled probe as a correction factor. This inventory provided a quantitative view of the steady-state level of each mRNA species. Genes the expression of which was detected by this method were enumerated, and results were compared with the current understanding of
E. coli
physiology.
Iron-Sulfur Flavoprotein (Isf) from
Methanosarcina thermophila
Is the Prototype of a Widely Distributed Family
Authors: Zhao Tong , Cruz Francisco , Ferry James G. 
Published: July 2002

ABSTRACT

A total of 35 homologs of the iron-sulfur flavoprotein (Isf) from
Methanosarcina thermophila
were identified in databases. All three domains were represented, and multiple homologs were present in several species. An unusually compact cysteine motif ligating the 4Fe-4S cluster in Isf is conserved in all of the homologs except two, in which either an aspartate or a histidine has replaced the second cysteine in the motif. A phylogenetic analysis of Isf homologs identified four subgroups, two of which were supported by bootstrap data. Three homologs from metabolically and phylogenetically diverse species in the
Bacteria
and
Archaea
domains (Af3 from
Archaeoglobus fulgidus
, Cd1 from
Clostridium difficile
, and Mj2 from
Methanococcus jannaschii
) were overproduced in
Escherichia coli
. Each homolog purified as a homodimer, and the UV-visible absorption spectra were nearly identical to that of Isf. After reconstitution with iron, sulfide, and flavin mononucleotide (FMN) the homologs contained six to eight nonheme iron atoms and 1.6 to 1.7 FMN molecules per dimer, suggesting that two 4Fe-4S or 3Fe-4S clusters and two FMN cofactors were bound to each dimer, which is consistent with Isf data. Homologs Af3 and Mj2 were reduced by CO in reactions catalyzed by cell extract of acetate-grown
M. thermophila
, but Cd1 was not. Homologs Af3 and Mj2 were reduced by CO in reactions catalyzed by
A. fulgidus
and
M. jannaschii
cell extracts. Cell extract of
Clostridium thermoaceticum
catalyzed CO reduction of Cd1. Our database sequence analyses and biochemical characterizations indicate that Isf is the prototype of a family of iron-sulfur flavoproteins that occur in members of all three domains.
relA
-Independent Amino Acid Starvation Response Network of
Streptococcus pyogenes
Authors: Steiner Kerstin , Malke Horst 
Published: July 2002

ABSTRACT

Streptococcus pyogenes
(group A streptococcus [GAS]), a multiple-amino-acid-auxotrophic human pathogen, may face starvation for essential amino acids during various stages of the infection process. Since the response of GAS to such conditions is likely to influence pathogenetic processes, we set out to identify by transcriptional analyses genes and operons that are responsive to amino acid starvation and examined whether functionally meaningful response patterns can be ascertained. We discovered that GAS are capable of mounting a
relA
-independent amino acid starvation response that involves transcriptional modulation of a wide array of housekeeping genes as well as accessory and dedicated virulence genes. Housekeeping genes that were upregulated during starvation of both wild-type and
relA
mutant strains included the newly identified T-box members of the aminoacyl-tRNA synthetase genes, the genes for components of the tmRNA-mediated peptide tagging and proteolysis system for abnormal proteins (
ssrA
,
smpB
,
clpP
, and
clpC
), and the operons for the
dnaK
and
groE
groups of molecular chaperones. In addition to upregulation of the genes for oligopeptide permease (
opp
), intracellular peptidase (
pepB
), and the two-component regulator
covRS
reported previously (K. Steiner and H. Malke, Mol. Microbiol. 38:1004–1016, 2000), amino acid starvation stimulated the transcription of the growth phase-associated, virulence-regulatory
fas
operon, the streptolysin S operon (
sag
), and the gene for autoinducer-2 production protein (
luxS
). A prominent feature of operons exhibiting internal transcriptional termination (
opp
,
fas
, and
sag
) was starvation-promoted full-length transcription, a mechanism that improves the efficacy of these systems by increasing the level of coordinate transcription of functionally related genes. Based on these results, a regulatory network with feedback mechanisms is proposed that counteracts the stringent response, links the levels of key rate-limiting enzymes to virulence gene expression, and enables the organism in a dynamic way to take advantage of protein-rich environments provided by its human host. As several of the affected target genes are controlled by more than one regulator, fine modulation may result in accordance with the demands imposed by ecologically different colonization sites upon the adaptive capacity of the pathogen.
Molecular Characterization ofDesulfovibrio gigasNeelaredoxin, a Protein Involved in Oxygen Detoxification in Anaerobes
Authors: Silva Gabriela , LeGall Jean , Xavier António V. , Teixeira Miguel , Rodrigues-Pousada Claudina 
Published: July 2002

ABSTRACTDesulfovibrio gigasneelaredoxin is an iron-containing protein of 15 kDa, having a single iron site with a His4Cys coordination. Neelaredoxins and homologous proteins are widespread in anaerobic prokaryotes and have superoxide-scavenging activity. To further understand its role in anaerobes, its genomic organization and expression inD. gigaswere studied and its ability to complementEscherichia colisuperoxide dismutase deletion mutant was assessed. InD. gigas, neelaredoxin is transcribed as a monocistronic mRNA of 500 bases as revealed by Northern analysis. Putative promoter elements resembling ς70recognition sequences were identified. Neelaredoxin is abundantly and constitutively expressed, and its expression is not further induced during treatment with O2or H2O2. The neelaredoxin gene was cloned by PCR and expressed inE. coli, and the protein was purified to homogeneity. The recombinant neelaredoxin has spectroscopic properties identical to those observed for the native one. Mutations of Cys-115, one of the iron ligands, show that this ligand is essential for the activity of neelaredoxin. In an attempt to elucidate the function of neelaredoxin within the cell, it was expressed in anE. colimutant deficient in cytoplasmic superoxide dismutases (sodA sodB). Neelaredoxin suppresses the deleterious effects produced by superoxide, indicating that it is involved in oxygen detoxification in the anaerobeD. gigas.
SecG Function and Phospholipid Metabolism in
Escherichia coli
Authors: Flower Ann M. 
Published: July 2002

ABSTRACT

SecG is an auxiliary protein in the Sec-dependent protein export pathway of
Escherichia coli
. Although the precise function of SecG is unknown, it stimulates translocation activity and has been postulated to enhance the membrane insertion-deinsertion cycle of SecA. Deletion of
secG
was initially reported to result in a severe export defect and cold sensitivity. Later results demonstrated that both of these phenotypes were strain dependent, and it was proposed that an additional mutation was required for manifestation of the cold-sensitive phenotype. The results presented here demonstrate that the cold-sensitive
secG
deletion strain also contains a mutation in
glpR
that causes constitutive expression of the
glp
regulon. Introduction of both the
glpR
mutation and the
secG
deletion into a wild-type strain background produced a cold-sensitive phenotype, confirming the hypothesis that a second mutation (
glpR
) contributes to the cold-sensitive phenotype of
secG
deletion strains. It was speculated that the
glpR
mutation causes an intracellular depletion of glycerol-3-phosphate due to constitutive synthesis of GlpD and subsequent channeling of glycerol-3-phosphate into metabolic pathways. In support of this hypothesis, it was demonstrated that addition of glycerol-3-phosphate to the growth medium ameliorated the cold sensitivity, as did introduction of a
glpD
mutation. This depletion of glycerol-3-phosphate is predicted to limit phospholipid biosynthesis, causing an imbalance in the levels of membrane phospholipids. It is hypothesized that this state of phospholipid imbalance imparts a dependence on SecG for proper function or stabilization of the translocation apparatus.
Endophytic Colonization of Rice by a Diazotrophic Strain of
Serratia marcescens
Authors: Gyaneshwar Prasad , James Euan K. , Mathan Natarajan , Reddy Pallavolu M. , Reinhold-Hurek Barbara , Ladha Jagdish K. 
Published: July 2002

ABSTRACT

Six closely related N
2
-fixing bacterial strains were isolated from surface-sterilized roots and stems of four different rice varieties. The strains were identified as
Serratia marcescens
by 16S rRNA gene analysis. One strain, IRBG500, chosen for further analysis showed acetylene reduction activity (ARA) only when inoculated into media containing low levels of fixed nitrogen (yeast extract). Diazotrophy of IRBG500 was confirmed by measurement of
15
N
2
incorporation and by sequence analysis of the PCR-amplified fragment of
nifH
. To examine its interaction with rice, strain IRBG500 was marked with
gusA
fused to a constitutive promoter, and the marked strain was inoculated onto rice seedlings under axenic conditions. At 3 days after inoculation, the roots showed blue staining, which was most intense at the points of lateral root emergence and at the root tip. At 6 days, the blue precipitate also appeared in the leaves and stems. More detailed studies using light and transmission electron microscopy combined with immunogold labeling confirmed that IRBG500 was endophytically established within roots, stems, and leaves. Large numbers of bacteria were observed within intercellular spaces, senescing root cortical cells, aerenchyma, and xylem vessels. They were not observed within intact host cells. Inoculation of IRBG500 resulted in a significant increase in root length and root dry weight but not in total N content of rice variety IR72. The inoculated plants showed ARA, but only when external carbon (e.g., malate, succinate, or sucrose) was added to the rooting medium.
Characterization of Outer Membrane Proteins in
Chlamydia trachomatis
LGV Serovar L2
Authors: Tanzer Regina J. , Hatch Thomas P. 
Published: July 2002

ABSTRACT

We used a photoactivatable, lipophilic reagent, 3′-(trifluoromethyl)-3-(
m
-[
125
I]iodophenyl)diazirine, to label proteins in the outer membrane of elementary bodies of
Chlamydia trachomatis
LGV serovar L2 and mass spectrometry to identify the labeled proteins. The identified proteins were polymorphic outer membrane proteins E, G, and H, which were made late in the developmental cycle, the major outer membrane protein, and a mixture of 46-kDa proteins consisting of the open reading frame 623 protein and possibly a modified form of the major outer membrane protein.