bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization ofVibrio choleraeO1 Antigen as the Bacteriophage K139 Receptor and Identification of IS1004Insertions Aborting O1 Antigen Biosynthesis
Authors: Nesper Jutta , Kapfhammer Dagmar , Klose Karl E. , Merkert Hilde , Reidl Joachim 
Published: July 2002

ABSTRACTBacteriophage K139 was recently characterized as a temperate phage of O1Vibrio cholerae. In this study we have determined the phage adsorption site on the bacterial cell surface. Phage-binding studies with purified lipopolysaccharide (LPS) of different O1 serotypes and biotypes revealed that the O1 antigen serves as the phage receptor. In addition, phage-resistant O1 El Tor strains were screened by using a virulent isolate of phage K139. Analysis of the LPS of such spontaneous phage-resistant mutants revealed that most of them synthesize incomplete LPS molecules, composed of either defective O1 antigen or core oligosaccharide. By applying phage-binding studies, it was possible to distinguish between receptor mutants and mutations which probably caused abortion of later steps of phage infection. Furthermore, we investigated the genetic nature of O1-negative strains by Southern hybridization with probes specific for the O antigen biosynthesis cluster (rfbregion). Two of the investigated O1 antigen-negative mutants revealed insertions of element IS1004into therfbgene cluster. Treating onewbeW::IS1004serum-sensitive mutant with normal human serum, we found that several survivors showed precise excision of IS1004, restoring O antigen biosynthesis and serum resistance. Investigation of clinical isolates by screening for phage resistance and performing LPS analysis of nonlysogenic strains led to the identification of a strain with decreased O1 antigen presentation. This strain had a significant reduction in its ability to colonize the mouse small intestine.
Purine Catabolism in
Escherichia coli
and Function of Xanthine Dehydrogenase in Purine Salvage
Authors: Xi Hualin , Schneider Barbara L. , Reitzer Larry 
Published: July 2002

ABSTRACT

Escherichia coli
is not known to utilize purines, other than adenine and adenosine, as nitrogen sources. We reinvestigated purine catabolism because a computer analysis suggested several potential ς
54
-dependent promoters within a 23-gene cluster whose products have homology to purine catabolic enzymes. Our results did not provide conclusive evidence that the ς
54
-dependent promoters are active. Nonetheless, our results suggest that some of the genes are metabolically significant. We found that even though several purines did not support growth as the sole nitrogen source, they did stimulate growth with aspartate as the nitrogen source. Cells produced
14
CO
2
from minimal medium containing [
14
C]adenine, which implies allantoin production. However, neither ammonia nor carbamoyl phosphate was produced, which implies that purine catabolism is incomplete and does not provide nitrogen during nitrogen-limited growth. We constructed strains with deletions of two genes whose products might catalyze the first reaction of purine catabolism. Deletion of one eliminated
14
CO
2
production from [
14
C]adenine, which implies that its product is necessary for xanthine dehydrogenase activity. We changed the name of this gene to
xdhA
. The
xdhA
mutant grew faster with aspartate as a nitrogen source. The mutant also exhibited sensitivity to adenine, which guanosine partially reversed. Adenine sensitivity has been previously associated with defective purine salvage resulting from impaired synthesis of guanine nucleotides from adenine. We propose that xanthine dehydrogenase contributes to this purine interconversion.
Regulation of
Vibrio cholerae
Genes Required for Acid Tolerance by a Member of the “ToxR-Like” Family of Transcriptional Regulators
Authors: Merrell D. Scott , Camilli Andrew 
Published: July 2002

ABSTRACT

The ability of the intestinal pathogen
Vibrio cholerae
to undergo an adaptive stress response, known as the acid tolerance response (ATR), was previously shown to enhance virulence. An essential component of the ATR is CadA-mediated lysine decarboxylation. CadA is encoded by the acid- and infection-induced gene
cadA
. Herein,
cadA
is shown to be the second gene in an operon with
cadB
, encoding a lysine/cadaverine antiporter.
cadC
, which is 5′ of
cadB
, encodes an acid-responsive, positive transcriptional regulator of
cadBA
. Unlike in
Escherichia coli
,
V. cholerae cadB
and
cadA
are also transcribed monocistronically. Of note, bicistronic
cadBA
is transcribed at low constitutive levels in an acid- and CadC-independent manner. CadC represents a new member of the “ToxR-like” family of transcriptional regulators in
V. cholerae
and, in addition, exhibits extensive amino acid and functional similarity to
E. coli
CadC. The amino-terminal, putative DNA binding domains of ToxR and CadC are highly conserved, as are the putative promoter elements recognized by these transcription factors.
Cloning and Characterization of Secretory Tyrosine Phosphatases of
Mycobacterium tuberculosis
Authors: Koul Anil , Choidas Axel , Treder Martin , Tyagi Anil K. , Drlica Karl , Singh Yogendra , Ullrich Axel 
Published: July 2002

ABSTRACT

Two genes with sequence homology to those encoding protein tyrosine phosphatases were cloned from genomic DNA of
Mycobacterium tuberculosis
H
37
Rv. The calculated molecular masses of these two putative tyrosine phosphatases, designated MPtpA and MPtpB, were 17.5 and 30 kDa, respectively. MPtpA and MPtpB were expressed as glutathione
S
-transferase fusion proteins in
Escherichia coli
. The affinity-purified proteins dephosphorylated the phosphotyrosine residue of myelin basic protein (MBP), but they failed to dephosphorylate serine/threonine residues of MBP. The activity of these phosphatases was inhibited by sodium orthovanadate, a specific inhibitor of tyrosine phosphatases, but not by okadaic acid, an inhibitor of serine/threonine phosphatases. Mutations at the catalytic site motif, cysteine 11 of MPtpA and cysteine 160 of MPtpB, abolished enzyme activity. Southern blot analysis revealed that, while
mptpA
is present in slow-growing mycobacterial species as well as fast-growing saprophytes,
mptpB
was restricted to members of the
M. tuberculosis
complex. These phosphatases were present in both whole-cell lysates and culture filtrates of
M. tuberculosis
, suggesting that these proteins are secreted into the extracellular medium. Since tyrosine phosphatases are essential for the virulence of several pathogenic bacteria, the restricted distribution of
mptpB
makes it a good candidate for a virulence gene of
M. tuberculosis
.
The Feedback Response of
Escherichia coli
rRNA Synthesis Is Not Identical to the Mechanism of Growth Rate-Dependent Control
Authors: Voulgaris Justina , Pokholok Dmitry , Holmes W. Mike , Squires Craig , Squires Catherine L. 
Published: July 2002

ABSTRACT

Growth rate-independent
rrn
P1 promoter mutants were tested for their ability to respond to changes in
rrn
gene dosage. Most were found to be normal for the feedback response. In addition, cellular levels of the initiating nucleoside triphosphates remained unchanged when the
rrn
gene dosage was altered. These results suggest that the feedback response cannot be the mechanism for growth rate-dependent control of rRNA synthesis and that the relationship between these two processes may be more complicated than is currently understood.
Inorganic Polyphosphate in
Vibrio cholerae
: Genetic, Biochemical, and Physiologic Features
Authors: Ogawa Nobuo , Tzeng Chi-Meng , Fraley Cresson D. , Kornberg Arthur 
Published: July 2002

ABSTRACT

Vibrio cholerae
O1, biotype El Tor, accumulates inorganic polyphosphate (poly P) principally as large clusters of granules. Poly P kinase (PPK), the enzyme that synthesizes poly P from ATP, is encoded by the
ppk
gene, which has been cloned from
V. cholerae
, overexpressed, and knocked out by insertion-deletion mutagenesis. The predicted amino acid sequence of PPK is 701 residues (81.6 kDa), with 64% identity to that of
Escherichia coli
, which it resembles biochemically. As in
E. coli
,
ppk
is part of an operon with
ppx
, the gene that encodes exopolyphosphatase (PPX). However, unlike in
E. coli
, PPX activity was not detected in cell extracts of wild-type
V. cholerae
. The
ppk
null mutant of
V. cholerae
has diminished adaptation to high concentrations of calcium in the medium as well as motility and abiotic surface attachment.
WhiD and WhiB, Homologous Proteins Required for Different Stages of Sporulation in
Streptomyces coelicolor
A3(2)
Authors: Molle Virginie , Palframan Wendy J. , Findlay Kim C. , Buttner Mark J. 
Published: July 2002

ABSTRACT

The
whiD
locus, which is required for the differentiation of
Streptomyces coelicolor
aerial hyphae into mature spore chains, was localized by map-based cloning to the overlap between cosmids 6G4 and D63 of the minimal ordered library of Redenbach et al. (M. Redenbach et al., Mol. Microbiol. 21:77–96, 1996). Subcloning and sequencing showed that
whiD
encodes a homologue of WhiB, a protein required for the initiation of sporulation septation in
S. coelicolor
. WhiD and WhiB belong to a growing family of small (76- to 112-residue) proteins of unknown biochemical function in which four cysteines are absolutely conserved; all known members of this family are found in the actinomycetes. A constructed
whiD
null mutant showed reduced levels of sporulation, and those spores that did form were heat sensitive, lysed extensively, and were highly irregular in size, arising at least in part from irregularity in septum placement. The
whiD
null mutant showed extreme variation in spore cell wall deposition; most spores had uniformly thin (20- to 30-nm) walls, but spore chains were frequently observed in which there was irregular but very pronounced (up to 170 nm) cell wall thickening at the junctions between spores.
whiD
null mutant spores were frequently partitioned into irregular smaller units through the deposition of additional septa, which were often laid down in several different planes, very close to the spore poles. These “minicompartments” appeared to be devoid of chromosomal DNA. Two
whiD
promoters,
whiDp1
and
whiDp2
, were identified, and their activities were analyzed during development of wild-type
S. coelicolor
on solid medium. Both promoters were developmentally regulated;
whiDp1
and
whiDp2
transcripts were detected transiently, approximately at the time when sporulation septa were observed in the aerial hyphae.
Regulation of rRNA Transcription Is Remarkably Robust: FIS Compensates for Altered Nucleoside Triphosphate Sensing by Mutant RNA Polymerases at
Escherichia coli rrn
P1 Promoters
Authors: Bartlett Michael S. , Gaal Tamas , Ross Wilma , Gourse Richard L. 
Published: July 2002

ABSTRACT

We recently identified
Escherichia coli
RNA polymerase (RNAP) mutants (RNAP β′ Δ215–220 and β RH454) that form extremely unstable complexes with rRNA P1 (
rrn
P1) core promoters. The mutant RNAPs reduce transcription and alter growth rate-dependent regulation of
rrn
P1 core promoters, because the mutant RNAPs require higher concentrations of the initiating nucleoside triphosphate (NTP) for efficient transcription from these promoters than are present in vivo. Nevertheless, the mutants grow almost as well as wild-type cells, suggesting that rRNA synthesis is not greatly perturbed. We report here that the
rrn
transcription factor FIS activates the mutant RNAPs more strongly than wild-type RNAP, thereby compensating for the altered properties of the mutant RNAPs. FIS activates the mutant RNAPs, at least in part, by reducing the apparent
K
ATP
for the initiating NTP. This and other results suggest that FIS affects a step in transcription initiation after closed-complex formation in addition to its stimulatory effect on initial RNAP binding. FIS and NTP levels increase with growth rate, suggesting that changing FIS concentrations, in conjunction with changing NTP concentrations, are responsible for growth rate-dependent regulation of
rrn
P1 transcription in the mutant strains. These results provide a dramatic demonstration of the interplay between regulatory mechanisms in rRNA transcription.
Regions of RNase E Important for 5′-End-Dependent RNA Cleavage and Autoregulated Synthesis
Authors: Jiang Xunqing , Diwa Alexis , Belasco Joel G. 
Published: July 2002

ABSTRACT

RNase E is an important regulatory enzyme that plays a key role in RNA processing and degradation in
Escherichia coli
. Internal cleavage by this endonuclease is accelerated by the presence of a monophosphate at the RNA 5′ end. Here we show that the preference of
E. coli
RNase E for 5′-monophosphorylated substrates is an intrinsic property of the catalytically active amino-terminal half of the enzyme and does not require the carboxy-terminal region. This property is shared by the related
E. coli
ribonuclease CafA (RNase G) and by a cyanobacterial RNase E homolog derived from
Synechocystis
, indicating that the 5′-end dependence of RNase E is a general characteristic of members of this ribonuclease family, including those from evolutionarily distant species. Although it is dispensable for 5′-end-dependent RNA cleavage, the carboxy-terminal half of RNase E significantly enhances the ability of this ribonuclease to autoregulate its synthesis in
E. coli
. Despite similarities in amino acid sequence and substrate specificity, CafA is unable to replace RNase E in sustaining
E. coli
cell growth or in regulating RNase E production, even when overproduced sixfold relative to wild-type RNase E levels.
Carbon Flux Distribution and Kinetics of Cellulose Fermentation in Steady-State Continuous Cultures of
Clostridium cellulolyticum
on a Chemically Defined Medium
Authors: Desvaux Mickaël , Guedon Emmanuel , Petitdemange Henri 
Published: July 2002

ABSTRACT

The metabolic characteristics of
Clostridium cellulolyticum
, a mesophilic cellulolytic nonruminal bacterium, were investigated and characterized kinetically for the fermentation of cellulose by using chemostat culture analysis. Since with
C. cellulolyticum
(i) the ATP/ADP ratio is lower than 1, (ii) the production of lactate at low specific growth rate (μ) is low, and (iii) there is a decrease of the NADH/NAD
+
ratio and
q
NADH produced
/
q
NADH used
ratio as the dilution rate (
D
) increases in carbon-limited conditions, the chemostats used were cellulose-limited continuously fed cultures. Under all conditions, ethanol and acetate were the main end products of catabolism. There was no shift from an acetate-ethanol fermentation to a lactate-ethanol fermentation as previously observed on cellobiose as μ increased (E. Guedon, S. Payot, M. Desvaux, and H. Petitdemange, J. Bacteriol. 181:3262–3269, 1999). The acetate/ethanol ratio was always higher than 1 but decreased with
D
. On cellulose, glucose 6-phosphate and glucose 1-phosphate are important branch points since the longer the soluble β-glucan uptake is, the more glucose 1-phosphate will be generated. The proportion of carbon flowing toward phosphoglucomutase remained constant (around 59.0%), while the carbon surplus was dissipated through exopolysaccharide and glycogen synthesis. The percentage of carbon metabolized via pyruvate-ferredoxin oxidoreductase decreased with
D
. Acetyl coenzyme A was mainly directed toward the acetate formation pathway, which represented a minimum of 27.1% of the carbon substrate. Yet the proportion of carbon directed through biosynthesis (i.e., biomass, extracellular proteins, and free amino acids) and ethanol increased with
D
, reaching 27.3 and 16.8%, respectively, at 0.083 h
−1
. Lactate and extracellular pyruvate remained low, representing up to 1.5 and 0.2%, respectively, of the original carbon uptake. The true growth yield obtained on cellulose was higher, [50.5 g of cells (mol of hexose eq)
−1
] than on cellobiose, a soluble cellodextrin [36.2 g of cells (mol of hexose eq)
−1
]. The rate of cellulose utilization depended on the solid retention time and was first order, with a rate constant of 0.05 h
−1
. Compared to cellobiose, substrate hydrolysis by cellulosome when bacteria are grown on cellulose fibers introduces an extra means for regulation of the entering carbon flow. This led to a lower μ, and so metabolism was not as distorted as previously observed with a soluble substrate. From these results,
C. cellulolyticum
appeared well adapted and even restricted to a cellulolytic lifestyle.
The LuxM Homologue VanM from
Vibrio anguillarum
Directs the Synthesis of
N
-(3-Hydroxyhexanoyl)homoserine Lactone and
N
-Hexanoylhomoserine Lactone
Authors: Milton Debra L. , Chalker Victoria J. , Kirke David , Hardman Andrea , Cámara Miguel , Williams Paul 
Published: July 2002

ABSTRACT

Vibrio anguillarum
, which causes terminal hemorrhagic septicemia in fish, was previously shown to possess a LuxRI-type quorum-sensing system (
vanRI
) and to produce
N
-(3-oxodecanoyl)homoserine lactone (3-oxo-C10-HSL). However, a
vanI
null mutant still activated
N
-acylhomoserine lactone (AHL) biosensors, indicating the presence of an additional quorum-sensing circuit in
V. anguillarum
. In this study, we have characterized this second system. Using high-pressure liquid chromatography in conjunction with mass spectrometry and chemical analysis, we identified two additional AHLs as
N
-hexanoylhomoserine lactone (C6-HSL) and
N
-(3-hydroxyhexanoyl)homoserine lactone (3-hydroxy-C6-HSL). Quantification of each AHL present in stationary-phase
V. anguillarum
spent culture supernatants indicated that 3-oxo-C10-HSL, 3-hydroxy-C6-HSL, and C6-HSL are present at approximately 8.5, 9.5, and 0.3 nM, respectively. Furthermore,
vanM
, the gene responsible for the synthesis of these AHLs, was characterized and shown to be homologous to the
luxL
and
luxM
genes, which are required for the production of
N
-(3-hydroxybutanoyl)homoserine lactone in
Vibrio harveyi
. However, resequencing of the
V. harveyi luxL/luxM
junction revealed a sequencing error present in the published sequence, which when corrected resulted in a single open reading frame (termed
luxM
). Downstream of
vanM
, we identified a homologue of
luxN
(
vanN
) that encodes a hybrid sensor kinase which forms part of a phosphorelay cascade involved in the regulation of bioluminescence in
V. harveyi
. A mutation in
vanM
abolished the production of C6-HSL and 3-hydroxy-C6-HSL. In addition, production of 3-oxo-C10-HSL was abolished in the
vanM
mutant, suggesting that 3-hydroxy-C6-HSL and C6-HSL regulate the production of 3-oxo-C10-HSL via
vanRI
. However, a
vanN
mutant displayed a wild-type AHL profile. Neither mutation affected either the production of proteases or virulence in a fish infection model. These data indicate that
V. anguillarum
possesses a hierarchical quorum sensing system consisting of regulatory elements homologous to those found in both
V. fischeri
(the LuxRI homologues VanRI) and
V. harveyi
(the LuxMN homologues, VanMN).
Sodium Ion-Driven Serine/Threonine Transport in
Porphyromonas gingivalis
Authors: Dashper S. G. , Brownfield L. , Slakeski N. , Zilm P. S. , Rogers A. H. , Reynolds E. C. 
Published: July 2002

ABSTRACT

Porphyromonas gingivalis
is an asaccharolytic, gram-negative bacterium that relies on the fermentation of amino acids for metabolic energy. When grown in continuous culture in complex medium containing 4 mM (each) free serine, threonine, and arginine,
P. gingivalis
assimilated mainly glutamate/glutamine, serine, threonine, aspartate/asparagine, and leucine in free and/or peptide form. Serine and threonine were assimilated in approximately equal amounts in free and peptide form. We characterized serine transport in this bacterium by measuring uptake of the radiolabeled amino acid in washed cells of
P. gingivalis
energized with a tetrapeptide not containing serine. Serine was transported by a single system with an affinity constant for transport (

K
t

) of 24 μM that was competitively inhibited by threonine. Serine transport was dependent on sodium ion concentration in the suspending buffer, and the addition of the ionophore gramicidin caused the inhibition of serine uptake. Together these data indicate that serine transport was sodium ion-motive force driven. A
P. gingivalis
gene potentially encoding a serine transporter was identified by sequence similarity to an
Escherichia coli
serine transporter (SstT). This
P. gingivalis
gene, designated
sstT,
was inactivated by insertion of a
Bacteroides
tetQ
gene, producing the mutant W50ST. The mutant was unable to transport serine, confirming the presence of a single serine transporter in this bacterium under these growth conditions. The transport of serine by
P. gingivalis
was dependent on the presence of free cysteine in the suspension buffer. Other reducing agents were unable to stimulate serine uptake. These data show that
P. gingivalis
assimilates free serine and threonine from culture media via a cysteine-activated, sodium ion-motive force-driven serine/threonine transporter.
Competence Modulation by the NADH Oxidase of
Streptococcus pneumoniae
Involves Signal Transduction
Authors: Echenique José R. , Trombe Marie C. 
Published: July 2002

ABSTRACT

Oxygen controls competence development in
Streptococcus pneumoniae
. Oxygen signaling involves the two-component signal transduction systems CiaRH and ComDE and the competence-stimulating peptide encoded by
comC
and processed by ComAB. We found that NADH oxidase (Nox) was required for optimal competence. Transcriptional analysis and genetic dissection showed that Nox was involved in post-transcriptional activation of the response regulator ComE and in the transcriptional control of
ciaRH
and
comCDE
. Thus, in
S. pneumoniae,
Nox, with O
2
as its secondary substrate, is part of the O
2
-signaling pathway.
HcwA, an Autolysin, Is Required for Heterocyst Maturation in
Anabaena
sp. Strain PCC 7120
Authors: Zhu Jinsong , Jäger Karin , Black Todd , Zarka Kelly , Koksharova Olga , Wolk C. Peter 
Published: July 2002

ABSTRACT

In many filamentous cyanobacteria, vegetative cells can differentiate into heterocysts, cells that are specialized for aerobic fixation of N
2
. Synthesis of the heterocyst envelope polysaccharide is dependent on the gene
hepA
in
Anabaena
sp. strain PCC 7120. In search of genes that are involved in the regulation of
hepA
, we transposon mutagenized strain DR1069, which bears a chromosomal
hepA
::
luxAB
fusion. One resulting mutant, designated HNL3, grows normally in medium with nitrate and shows poor induction of
hepA
in response to nitrogen deprivation. In HNL3, transposon Tn
5-
1058 is inserted within gene
hcwA
, a constitutively expressed open reading frame whose predicted product resembles
N
-acetylmuramoyl-
l
-alanine amidases. Reconstruction of the mutation confirmed that the mutant phenotype resulted from the insertion of the transposon. The induction of
hepA
in HNL3 is partially restored upon recombination of HNL3 with plasmid-borne, wild-type
hcwA
. Moreover, HcwA expressed in
Escherichia coli
exhibits wall-lytic activity. These results suggest that the degradation, or possibly reconstruction, of the cell peptidoglycan layer is a prerequisite for heterocyst maturation.