bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transcription of Clumping Factor A in Attached and Unattached
Staphylococcus aureus
In Vitro and during Device-Related Infection
Authors: Wolz Christiane , Goerke Christiane , Landmann Regine , Zimmerli Werner , Fluckiger Ursula 
Published: July 2002

ABSTRACT

Staphylococcus aureus
is one of the pathogens most frequently isolated in device-related infections.
S. aureus
is equipped with surface-associated proteins promoting specific binding to matrix molecules. Clumping factor A (ClfA, encoded by
clfA
) mediates adhesion to fibrinogen. Whereas the contribution of ClfA to pathogenicity is well documented, the influence of different growth and host parameters on gene activity is unclear. To elucidate this question, we investigated
clfA
transcript levels in an animal model of device-related infection and in planktonic and sessile bacteria grown in vitro. Specific mRNA from the
S. aureus
strains Newman, Reynolds, and RN6390 was quantified by LightCycler reverse transcription-PCR. In vitro,
clfA
transcript levels were low in the early logarithmic growth phase, but a clear increase was observed after the late logarithmic phase. Quantities of
clfA
transcripts were four to six times higher in the planktonic than in the sessile bacterial subpopulations grown to the stationary phase. During infection, in strains Newman and Reynolds levels of
clfA
transcripts in exudates accumulating in the infected devices were lower than those in the bacteria grown in vitro to stationary phase.
clfA
mRNA levels in the exudates increased during the initial phase of infection and remained constant after 96 h postinoculation. In contrast to the in vitro results, quantities of
clfA
transcripts in the unattached bacteria of the exudates never exceeded the level of
clfA
transcripts in the sessile bacteria attached to glass beads. However, a clear increase in
clfA
quantities in the sessile bacteria was observed late in infection after 144 h. In conclusion, maximal
clfA
transcript levels are reached late during growth in vitro and in vivo.
Correlation of ESAT-6-Specific Gamma Interferon Production with Pathology in Cattle followingMycobacterium bovisBCG Vaccination against Experimental Bovine Tuberculosis
Authors: Vordermeier H. Martin , Chambers Mark A. , Cockle Paul J. , Whelan Adam O. , Simmons Jennifer , Hewinson R. Glyn 
Published: July 2002

ABSTRACTVaccine development and the understanding of the pathology of bovine tuberculosis in cattle would be greatly facilitated by the definition of immunological correlates of protection and/or pathology. To address these questions, cattle were vaccinated withMycobacterium bovisbacillus Calmette-Guérin (BCG) and were then challenged with virulentM. bovis. Applying a semiquantitative pathology-scoring system, we were able to demonstrate that BCG vaccination imparted significant protection by reducing the disease severity on average by 75%. Analysis of cellular immune responses followingM. bovischallenge demonstrated that proliferative T-cell and gamma interferon (IFN-γ) responses towards theM. bovis-specific antigen ESAT-6, whose gene is absent from BCG, were generally low in vaccinated animals but were high in all nonvaccinated calves. Importantly, the amount of ESAT-6-specific IFN-γ measured by enzyme-linked immunosorbent assay afterM. bovischallenge, but not the frequency of responding cells, correlated positively with the degree of pathology found 18 weeks after infection. Diagnostic reagents based on antigens not present in BCG, like ESAT-6 and CFP-10, were still able to distinguish BCG-vaccinated, diseased animals from BCG-vaccinated animals without signs of disease. In summary, our results suggest that the determination of ESAT-6-specific IFN-γ, while not a direct correlate of protection, constitutes nevertheless a useful prognostic immunological marker predicting both vaccine efficacy and disease severity.
Differential Activation of the
tcpPH
Promoter by AphB Determines Biotype Specificity of Virulence Gene Expression in
Vibrio cholerae
Authors: Kovacikova Gabriela , Skorupski Karen 
Published: July 2002

ABSTRACT

Vibrio cholerae
strains of the classical biotype express the genes encoding cholera toxin (CT) and toxin-coregulated pilus (TCP) under a variety of environmental conditions in vitro, whereas El Tor biotype strains express these genes only under specialized culture conditions. We show here that a single base-pair difference at positions −65 and −66 of the classical and El Tor
tcpPH
promoters, respectively, is responsible for the differential regulation of virulence gene expression in these two disease-causing biotypes. Analysis of
tcpP-lacZ
fusions in both
V. cholerae
and
Escherichia coli
indicated that transcriptional activation of the El Tor
tcpPH
promoter by the LysR regulator AphB was significantly reduced relative to that of the classical promoter. Reciprocal exchange of the
tcpPH
promoter between the two biotypes in
V. cholerae
showed that the ability to activate the transcription of
tcpPH
is not dependent on the biotype of the strain per se but on the
tcpPH
promoter itself. Classical and El Tor
tcpP-lacZ
promoter chimeras in
E. coli
localized the region responsible for the differential activation of
tcpPH
by AphB to within 75 bp of the transcriptional start site. Individual base-pair changes within this region showed that the presence of either an A or a G at position −65 or −66 conferred the classical or El Tor, respectively, pattern of
tcpPH
activation by AphB. Reciprocal exchange of these base pairs between biotypes in
V. cholerae
switched the biotype-specific pattern of expression of
tcpPH
as well as the production of CT and TCP in response to environmental stimuli.
Exopolysaccharide Production Is Required for Development of
Escherichia coli
K-12 Biofilm Architecture
Authors: Danese Paul N. , Pratt Leslie A. , Kolter Roberto 
Published: July 2002

ABSTRACT

Although exopolysaccharides (EPSs) are a large component of bacterial biofilms, their contribution to biofilm structure and function has been examined for only a few organisms. In each of these cases EPS has been shown to be required for cellular attachment to abiotic surfaces. Here, we undertook a genetic approach to examine the potential role of colanic acid, an EPS of
Escherichia coli
K-12, in biofilm formation. Strains either proficient or deficient in colanic acid production were grown and allowed to adhere to abiotic surfaces and were then examined both macroscopically and microscopically. Surprisingly, we found that colanic acid production is not required for surface attachment. Rather, colanic acid is critical for the formation of the complex three-dimensional structure and depth of
E. coli
biofilms.
Identification of
Escherichia coli ubiB
, a Gene Required for the First Monooxygenase Step in Ubiquinone Biosynthesis
Authors: Poon Wayne W. , Davis Diana E. , Ha Huan T. , Jonassen Tanya , Rather Philip N. , Clarke Catherine F. 
Published: July 2002

ABSTRACT

It was recently discovered that the
aarF
gene in
Providencia stuartii
is required for coenzyme Q (CoQ) biosynthesis. Here we report that
yigR
, the
Escherichia coli
homologue of
aarF
, is
ubiB
, a gene required for the first monooxygenase step in CoQ biosynthesis. Both the
P. stuartii aarF
and
E. coli ubiB
(
yigR
) disruption mutant strains lack CoQ and accumulate octaprenylphenol. Octaprenylphenol is the CoQ biosynthetic intermediate found to accumulate in the
E. coli
strain AN59, which contains the
ubiB409
mutant allele. Analysis of the mutation in the
E. coli
strain AN59 reveals no mutations within the
ubiB
gene, but instead shows the presence of an IS
1
element at position +516 of the
ubiE
gene. The
ubiE
gene encodes a
C
-methyltransferase required for the synthesis of both CoQ and menaquinone, and it is the 5′ gene in an operon containing
ubiE
,
yigP
, and
ubiB
. The data indicate that octaprenylphenol accumulates in AN59 as a result of a polar effect of the
ubiE
::IS
1
mutation on the downstream
ubiB
gene. AN59 is complemented by a DNA segment containing the contiguous
ubiE
,
yigP
, and
ubiB
genes. Although transformation of AN59 with a DNA segment containing the
ubiB
coding region fails to restore CoQ biosynthesis, transformation with the
ubiE
coding region results in a low-frequency but significant rescue attributed to homologous recombination. In addition, the
fre
gene, previously considered to correspond to
ubiB
, was found not to be involved in CoQ biosynthesis. The
ubiB
gene is a member of a predicted protein kinase family of which the
Saccharomyces cerevisiae ABC1
gene is the prototypic member. The possible protein kinase function of UbiB and Abc1 and the role these polypeptides may play in CoQ biosynthesis are discussed.
Structural and Kinetic Characterization of an Archaeal β-Class Carbonic Anhydrase
Authors: Smith Kerry S. , Cosper Nathaniel J. , Stalhandske Christina , Scott Robert A. , Ferry James G. 
Published: July 2002

ABSTRACT

The β-class carbonic anhydrase from the archaeon
Methanobacterium thermoautotrophicum
(Cab) was structurally and kinetically characterized. Analytical ultracentrifugation experiments show that Cab is a tetramer. Circular dichroism studies of Cab and the
Spinacia oleracea
(spinach) β-class carbonic anhydrase indicate that the secondary structure of the β-class enzymes is predominantly α-helical, unlike that of the α- or γ-class enzymes. Extended X-ray absorption fine structure results indicate the active zinc site of Cab is coordinated by two sulfur and two O/N ligands, with the possibility that one of the O/N ligands is derived from histidine and the other from water. Both the steady-state parameters
k
cat
and
k
cat
/

K
m

for CO
2
hydration are pH dependent. The steady-state parameter
k
cat
is buffer-dependent in a saturable manner at both pH 8.5 and 6.5, and the analysis suggested a ping-pong mechanism in which buffer is the second substrate. At saturating buffer conditions and pH 8.5,
k
cat
is 2.1-fold higher in H
2
O than in D
2
O, consistent with an intramolecular proton transfer step being rate contributing. The steady-state parameter
k
cat
/

K
m

is not dependent on buffer, and no solvent hydrogen isotope effect was observed. The results suggest a zinc hydroxide mechanism for Cab. The overall results indicate that prokaryotic β-class carbonic anhydrases have fundamental characteristics similar to the eukaryotic β-class enzymes and firmly establish that the α-, β-, and γ-classes are convergently evolved enzymes that, although structurally distinct, are functionally equivalent.
Identification of Genes in the RosR Regulon of
Rhizobium etli
Authors: Bittinger Mark A. , Handelsman Jo 
Published: July 2002

ABSTRACT

RosR is a determinant of nodulation competitiveness and cell surface characteristics of
Rhizobium etli
and has sequence similarity to a family of transcriptional repressors. To understand how RosR affects these phenotypes, we mutagenized a
rosR
mutant derivative of
R. etli
strain CE3 with a mini-Tn
5
that contains a promoterless
gusA
gene at one end, which acts as a transcriptional reporter. Using a mass-mating technique, we introduced
rosR
into each mutant in
trans
and screened for mutants that expressed different levels of β-glucuronidase activity in the presence and absence of
rosR
. A screen of 18,000 mutants identified 52 insertions in genes negatively regulated by RosR and 1 insertion in a gene positively regulated by RosR. Nucleotide sequence analysis of the regions flanking the insertions suggests that RosR regulates genes of diverse function, including those involved in polysaccharide production and in carbohydrate metabolism and those in a region containing sequence similarity to
virC1
and
virD3
from
Agrobacterium tumefaciens
. Two of the mutants produced colonies with altered morphology and were more competitive in nodulation than was CE3Δ
rosR
, the
rosR
parent. One mutant that contained an insertion in a gene with similarity to
exsH
of
Sinorhizobium meliloti
did not nodulate the plant host
Phaseolus vulgaris
without
rosR
. These results indicate that RosR directly or indirectly influences expression of diverse genes in
R. etli
, some of which affect the cell surface and nodulation competitiveness.
Pyruvate Kinase of the Hyperthermophilic Crenarchaeote
Thermoproteus tenax
: Physiological Role and Phylogenetic Aspects
Authors: Schramm Alexander , Siebers Bettina , Tjaden Britta , Brinkmann Henner , Hensel Reinhard 
Published: July 2002

ABSTRACT

Pyruvate kinase (PK; EC
2.7.1.40
) of
Thermoproteus tenax
was purified to homogeneity, and its coding gene was cloned and expressed in
Escherichia coli
. It represents a homomeric tetramer with a molecular mass of 49 kDa per subunit. PK exhibits positive binding cooperativity with respect to phosphoenolpyruvate and metal ions such as Mg
2+
and Mn
2+
. Heterotropic effects, as commonly found for PKs from bacterial and eucaryal sources, could not be detected. The enzyme does not depend on K
+
ions. Heterotrophically grown cells exhibit specific activity of PK four times higher than autotrophically grown cells. Since the mRNA level of the PK coding gene is also accordingly higher in heterotrophic cells, we conclude that the PK activity is adjusted to growth conditions mainly on the transcript level. The enzymic properties of the PK and the regulation of its expression are discussed with respect to the physiological framework given by the
T. tenax
-specific variant of the Embden-Meyerhof-Parnas pathway.
T. tenax
PK shows moderate overall sequence similarity (25 to 40% identity) to its bacterial and eucaryal pendants. Phylogenetic analyses of the known PK sequences result in a dichotomic tree topology that divides the enzymes into two major PK clusters, probably diverged by an early gene duplication event. The phylogenetic divergence is paralleled by a striking phenotypic differentiation of PKs: PKs of cluster I, which occur in eucaryal cytoplasm, some gamma proteobacteria, and low-GC gram-positive bacteria, are only active in the presence of fructose-1,6-bisphosphate or other phosphorylated sugars, whereas PKs of cluster II, found in various bacterial phyla, plastids, and in
Archaea
, show activity without effectors but are commonly regulated by the energy charge of the cell.
Role of Ger Proteins in Nutrient and Nonnutrient Triggering of Spore Germination in
Bacillus subtilis
Authors: Paidhungat Madan , Setlow Peter 
Published: July 2002

ABSTRACT

Dormant
Bacillus subtilis
spores germinate in the presence of particular nutrients called germinants. The spores are thought to recognize germinants through receptor proteins encoded by the
gerA
family of operons, which includes
gerA
,
gerB
, and
gerK
. We sought to substantiate this putative function of the GerA family proteins by characterizing spore germination in a mutant strain that contained deletions at all known
gerA
-like loci. As expected, the mutant spores germinated very poorly in a variety of rich media. In contrast, they germinated like wild-type spores in a chemical germinant, a 1-1 chelate of Ca
2+
and dipicolinic acid (DPA). These observations showed that proteins encoded by
gerA
family members are required for nutrient-induced germination but not for chemical-triggered germination, supporting the hypothesis that the GerA family encodes receptors for nutrient germinants. Further characterization of Ca
2+
–DPA-induced germination showed that the effect of Ca
2+
–DPA on spore germination was saturated at 60 mM and had a

K
m

of 30 mM. We also found that decoating spores abolished their ability to germinate in Ca
2+
–DPA but not in nutrient germinants, indicating that Ca
2+
–DPA and nutrient germinants probably act through parallel arms of the germination pathway.
Activation by Gene Amplification of
pitB
, Encoding a Third Phosphate Transporter of
Escherichia coli
K-12
Authors: Hoffer Sally M. , Schoondermark Paul , van Veen Hendrik W. , Tommassen Jan 
Published: July 2002

ABSTRACT

Two systems for the uptake of inorganic phosphate (P
i
) in
Escherichia coli
, PitA and Pst, have been described. A revertant of a
pitA pstS
double mutant that could grow on P
i
was isolated. We demonstrate that the expression of a new P
i
transporter, PitB, is activated in this strain by a gene amplification event.
Dual Repression by Fe
2+
-Fur and Mn
2+
-MntR of the
mntH
Gene, Encoding an NRAMP-Like Mn
2+
Transporter in
Escherichia coli
Authors: Patzer Silke I. , Hantke Klaus 
Published: July 2002

ABSTRACT

The uptake of Mn
2+
, a cofactor for several enzymes in
Escherichia coli
, is mediated by MntH, a proton-dependent metal transporter, which also recognizes Fe
2+
with lower affinity. MntH belongs to the NRAMP family of eukaryotic Fe
2+
and Mn
2+
transporters. In
E. coli
strains with chromosomal
mntH-lacZ
fusions,
mntH
was partially repressed by both Mn
2+
and Fe
2+
. Inactivation of
fur
resulted in the loss of Fe
2+
-dependent repression of
mntH
transcription, demonstrating that Fe
2+
repression depends on the global iron regulator Fur. However, these
fur
mutants still showed Mn
2+
-dependent repression of
mntH
. The Mn
2+
-responsive transcriptional regulator of
mntH
was identified as the gene product of
o155
(renamed MntR).
mntR
mutants were impaired in Mn
2+
but not Fe
2+
repression of
mntH
transcription. Binding of purified MntR to the
mntH
operator was manganese dependent. The binding region was localized by DNase I footprinting analysis and covers a nearly perfect palindrome. The Fur binding site, localized within 22 nucleotides of the
mntH
operator by in vivo operator titration assays, resembles the Fur-box consensus sequence.
pbpB
, a Gene Coding for a Putative Penicillin-Binding Protein, Is Required for Aerobic Nitrogen Fixation in the Cyanobacterium
Anabaena
sp. Strain PCC7120
Authors: Lázaro Sara , Fernández-Piñas Francisca , Fernández-Valiente Eduardo , Blanco-Rivero Amaya , Leganés Francisco 
Published: July 2002

ABSTRACT

Transposon mutagenesis of
Anabaena
sp. strain PCC7120 led to the isolation of a mutant strain, SNa1, which is unable to fix nitrogen aerobically but is perfectly able to grow with combined nitrogen (i.e., nitrate). Reconstruction of the transposon mutation of SNa1 in the wild-type strain reproduced the phenotype of the original mutant. The transposon had inserted within an open reading frame whose translation product shows significant homology with a family of proteins known as high-molecular-weight penicillin-binding proteins (PBPs), which are involved in the synthesis of the peptidoglycan layer of the cell wall. A sequence similarity search allowed us to identify at least 12 putative PBPs in the recently sequenced
Anabaena
sp. strain PCC7120 genome, which we have named and organized according to predicted molecular size and the
Escherichia coli
nomenclature for PBPs; based on this nomenclature, we have denoted the gene interrupted in SNal as
pbpB
and its product as PBP2. The wild-type form of
pbpB
on a shuttle vector successfully complemented the mutation in SNa1. In vivo expression studies indicated that PBP2 is probably present when both sources of nitrogen, nitrate and N
2
, are used. When nitrate is used, the function of PBP2 either is dispensable or may be substituted by other PBPs; however, under nitrogen deprivation, where the differentiation of the heterocyst takes place, the role of PBP2 in the formation and/or maintenance of the peptidoglycan layer is essential.
Hydrogen Peroxide Fluxes and Compartmentalization inside Growing
Escherichia coli
Authors: Seaver Lauren Costa , Imlay James A. 
Published: July 2002

ABSTRACT

Escherichia coli
generates about 14 μM hydrogen peroxide (H
2
O
2
) per s when it grows exponentially in glucose medium. The steady-state intracellular concentration of H
2
O
2
depends on the rates at which this H
2
O
2
is dissipated by scavenging enzymes and by efflux from the cell. The rates of H
2
O
2
degradation by the two major scavenging enzymes, alkyl hydroperoxide reductase and catalase, were quantified. In order to estimate the rate of efflux, the permeability coefficient of membranes for H
2
O
2
was determined. The coefficient is 1.6 × 10
−3
cm/s, indicating that permeability is substantial but not unlimited. These data allowed internal H
2
O
2
fluxes and concentrations to be calculated. Under these growth conditions, Ahp scavenges the majority of the endogenous H
2
O
2
, with a small fraction degraded by catalase and virtually none persisting long enough to penetrate the membrane and exit the cell. The robust scavenging activity maintains the H
2
O
2
concentration inside glucose-grown cells at <10
−7
M, substantially below the level (10
−6
M) at which toxicity is evident. When extracellular H
2
O
2
is present, its flux into the cell can be rapid, but the internal concentration may still be an order of magnitude lower than that outside. The presence of such gradients was confirmed in experiments that revealed different degrees of oxidative stress in cocultured scavenger-deficient mutants. The limited permeability of membranes to H
2
O
2
rationalizes the compartmentalization of scavenging systems and predicts that bacteria that excrete redox-cycling drugs do not experience the same H
2
O
2
dose that they impose on their competitors.
PatS and Products of Nitrogen Fixation Control Heterocyst Pattern
Authors: Yoon Ho-Sung , Golden James W. 
Published: July 2002

ABSTRACT

The filamentous cyanobacterium
Anabaena
sp. strain PCC 7120 forms a developmental pattern of single heterocysts separated by approximately 10 vegetative cells. Heterocysts differentiate from vegetative cells and are specialized for nitrogen fixation. The
patS
gene, which encodes a small peptide that inhibits heterocyst differentiation, is expressed in proheterocysts and plays a critical role in establishing the heterocyst pattern. Here we present further analysis of
patS
expression and heterocyst pattern formation. A
patS
-
gfp
reporter strain revealed clusters of
patS
-expressing cells during the early stage of heterocyst differentiation. PatS signaling is likely to be involved in the resolution of these clusters. Differentiating cells were inhibited by PatS during the time period 6 to 12 h after heterocyst induction, when groups of differentiating cells were being resolved to a single proheterocyst. Increased transcription of
patS
during development coincided with expression from a new transcription start site. In vegetative cells grown on nitrate, the 5′ end of a transcript for
patS
was localized 314 bases upstream from the first translation initiation codon. After heterocyst induction, a new transcript with a 5′ end at −39 bases replaced the vegetative cell transcript. A
patS
mutant grown for several days under nitrogen-fixing conditions showed partial restoration of the normal heterocyst pattern, presumably because of a gradient of nitrogen compounds supplied by the heterocysts. The
patS
mutant formed heterocysts when grown in the presence of nitrate but showed no nitrogenase activity and no obvious heterocyst pattern. We conclude that PatS and products of nitrogen fixation are the main signals determining the heterocyst pattern.