bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Mammalian Neuroendocrine Hormone Norepinephrine Supplies Iron for Bacterial Growth in the Presence of Transferrin or Lactoferrin
Authors: Freestone Primrose P. E. , Lyte Mark , Neal Christopher P. , Maggs Anthony F. , Haigh Richard D. , Williams Peter H. 
Published: July 2002

ABSTRACT

Norepinephrine stimulates the growth of a range of bacterial species in nutritionally poor SAPI minimal salts medium containing 30% serum. Addition of size-fractionated serum components to SAPI medium indicated that transferrin was required for norepinephrine stimulation of growth of
Escherichia coli
. Since bacteriostasis by serum is primarily due to the iron-withholding capacity of transferrin, we considered the possibility that norepinephrine can overcome this effect by supplying transferrin-bound iron for growth. Incubation with concentrations of norepinephrine that stimulated bacterial growth in serum-SAPI medium resulted in loss of bound iron from iron-saturated transferrin, as indicated by the appearance of monoferric and apo- isoforms upon electrophoresis in denaturing gels. Norepinephrine also caused the loss of iron from lactoferrin. The pharmacologically inactive metabolite norepinephrine 3-
O
-sulfate, by contrast, did not result in iron loss from transferrin or lactoferrin and did not stimulate bacterial growth in serum-SAPI medium. Norepinephrine formed stable complexes with transferrin, lactoferrin, and serum albumin. Norepinephrine-transferrin and norepinephrine-lactoferrin complexes, but not norepinephrine-apotransferrin or norepinephrine-albumin complexes, stimulated bacterial growth in serum-SAPI medium in the absence of additional norepinephrine. Norepinephrine-stimulated growth in medium containing
55
Fe complexed with transferrin or lactoferrin resulted in uptake of radioactivity by bacterial cells. Moreover, norepinephrine-stimulated growth in medium containing [
3
H]norepinephrine indicated concomitant uptake of norepinephrine. In each case, addition of excess iron did not affect growth but significantly reduced levels of radioactivity (
55
Fe or
3
H) associated with bacterial cells. A role for catecholamine-mediated iron supply in the pathophysiology of infectious diseases is proposed.
Spore Photoproduct (SP) Lyase from
Bacillus subtilis
Specifically Binds to and Cleaves SP (5-Thyminyl-5,6-Dihydrothymine) but Not Cyclobutane Pyrimidine Dimers in UV-Irradiated DNA
Authors: Slieman Tony A. , Rebeil Roberto , Nicholson Wayne L. 
Published: July 2002

ABSTRACT

The predominant photolesion in the DNA of UV-irradiated dormant bacterial spores is the thymine dimer 5-thyminyl-5,6-dihydrothymine, commonly referred to as spore photoproduct (SP). A major determinant of SP repair during spore germination is its direct reversal by the enzyme SP lyase, encoded by the
splB
gene in
Bacillus subtilis
. SplB protein containing an N-terminal tag of six histidine residues [(6His)SplB] was purified from dormant
B. subtilis
spores and shown to efficiently cleave SP but not cyclobutane
cis
,
syn
thymine-thymine dimers in vitro. In contrast, SplB protein containing an N-terminal 10-histidine tag [(10His)SplB] purified from an
Escherichia coli
overexpression system was incompetent to cleave SP unless the 10-His tag was first removed by proteolysis at an engineered factor Xa site. To assay the parameters of binding of SplB protein to UV-damaged DNA, a 35-bp double-stranded oligonucleotide was constructed which carried a single pair of adjacent thymines on one strand. Irradiation of the oligonucleotide in aqueous solution or at 10% relative humidity resulted in formation of cyclobutane pyrimidine dimers (Py◊Py) or SP, respectively. (10His)SplB was assayed for oligonucleotide binding using a DNase I protection assay. In the presence of (10His)SplB, the SP-containing oligonucleotide was selectively protected from DNase I digestion (half-life, >60 min), while the Py◊Py-containing oligonucleotide and the unirradiated oligonucleotide were rapidly digested by DNase I (half-lives, 6 and 9 min, respectively). DNase I footprinting of (10His)SplB bound to the artificial substrate was carried out utilizing the
32
P end-labeled 35-bp oligonucleotide containing SP. DNase I footprinting showed that SplB protected at least a 9-bp region surrounding SP from digestion with DNase I with the exception of two DNase I-hypersensitive sites within the protected region. (10His)SplB also caused significant enhancement of DNase I digestion of the SP-containing oligonucleotide for at least a full helical turn 3′ to the protected region. The data suggest that binding of SP lyase to SP causes significant bending or distortion of the DNA helix in the vicinity of the lesion.
The Spirochete FlaA Periplasmic Flagellar Sheath Protein Impacts Flagellar Helicity
Authors: Li Chunhao , Corum Linda , Morgan David , Rosey Everett L. , Stanton Thaddeus B. , Charon Nyles W. 
Published: July 2002

ABSTRACT

Spirochete periplasmic flagella (PFs), including those from
Brachyspira
(
Serpulina
),
Spirochaeta
,
Treponema
, and
Leptospira
spp., have a unique structure. In most spirochete species, the periplasmic flagellar filaments consist of a core of at least three proteins (FlaB1, FlaB2, and FlaB3) and a sheath protein (FlaA). Each of these proteins is encoded by a separate gene. Using
Brachyspira hyodysenteriae
as a model system for analyzing PF function by allelic exchange mutagenesis, we analyzed purified PFs from previously constructed
flaA
::
cat
,
flaA
::
kan
, and
flaB1
::
kan
mutants and newly constructed
flaB2
::
cat
and
flaB3
::
cat
mutants. We investigated whether any of these mutants had a loss of motility and altered PF structure. As formerly found with
flaA
::
cat
,
flaA
::
kan
, and
flaB1
::
kan
mutants,
flaB2
::
cat
and
flaB3
::
cat
mutants were still motile, but all were less motile than the wild-type strain, using a swarm-plate assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis indicated that each mutation resulted in the specific loss of the cognate gene product in the assembled purified PFs. Consistent with these results, Northern blot analysis indicated that each flagellar filament gene was monocistronic. In contrast to previous results that analyzed PFs attached to disrupted cells, purified PFs from a
flaA
::
cat
mutant were significantly thinner (19.6 nm) than those of the wild-type strain and
flaB1
::
kan
,
flaB2
::
cat
, and
flaB3
::
cat
mutants (24 to 25 nm). These results provide supportive genetic evidence that FlaA forms a sheath around the FlaB core. Using high-magnification dark-field microscopy, we also found that
flaA
::
cat
and
flaA
::
kan
mutants produced PFs with a smaller helix pitch and helix diameter compared to the wild-type strain and
flaB
mutants. These results indicate that the interaction of FlaA with the FlaB core impacts periplasmic flagellar helical morphology.
Ralstonia eutropha
TF93 Is Blocked in Tat-Mediated Protein Export
Authors: Bernhard Michael , Friedrich Bärbel , Siddiqui Roman A. 
Published: July 2002

ABSTRACT

Ralstonia eutropha
(formerly
Alcaligenes eutrophus
) TF93 is pleiotropically affected in the translocation of redox enzymes synthesized with an N-terminal signal peptide bearing a twin arginine (S/T-R-R-X-F-L-K) motif. Immunoblot analyses showed that the catalytic subunits of the membrane-bound [NiFe] hydrogenase (MBH) and the molybdenum cofactor-binding periplasmic nitrate reductase (Nap) are mislocalized to the cytoplasm and to the inner membrane, respectively. Moreover, physiological studies showed that the copper-containing nitrous oxide reductase (NosZ) was also not translocated to the periplasm in strain TF93. The cellular localization of enzymes exported by the general secretion system was unaffected. The translocation-arrested MBH and Nap proteins were enzymatically active, suggesting that twin-arginine signal peptide-dependent redox enzymes may have their cofactors inserted prior to transmembrane export. The periplasmic destination of MBH, Nap, and NosZ was restored by heterologous expression of
Azotobacter chroococcum tatA
mobilized into TF93.
tatA
encodes a bacterial Hcf106-like protein, a component of a novel protein transport system that has been characterized in thylakoids and shown to translocate folded proteins across the membrane.
Succinate:Quinol Oxidoreductases in the Cyanobacterium
Synechocystis
sp. Strain PCC 6803: Presence and Function in Metabolism and Electron Transport
Authors: Cooley Jason W. , Howitt Crispin A. , Vermaas Wim F. J. 
Published: July 2002

ABSTRACT

The open reading frames
sll
1625 and
sll
0823, which have significant sequence similarity to genes coding for the FeS subunits of succinate dehydrogenase and fumarate reductase, were deleted singly and in combination in the cyanobacterium
Synechocystis
sp. strain PCC 6803. When the organic acid content in the Δ
sll
1625 and Δ
sll
0823 strains was analyzed, a 100-fold decrease in succinate and fumarate concentrations was observed relative to the wild type. A similar analysis for the Δ
sll
1625 Δ
sll
0823 strain revealed that 17% of the wild-type succinate levels remained, while only 1 to 2% of the wild-type fumarate levels were present. Addition of 2-oxoglutarate to the growth media of the double mutant strain prior to analysis of organic acids in cells caused succinate to accumulate. This indicates that succinate dehydrogenase activity had been blocked by the deletions and that 2-oxoglutarate can be converted to succinate in vivo in this organism, even though a traditional 2-oxoglutarate dehydrogenase is lacking. In addition, reduction of the thylakoid plastoquinone pool in darkness in the presence of KCN was up to fivefold slower in the mutants than in the wild type. Moreover, in vitro succinate dehydrogenase activity observed in wild-type membranes is absent from those isolated from the double mutant and reduced in those from the single mutants, further indicating that the
sll
1625 and
sll
0823 open reading frames encode subunits of succinate dehydrogenase complexes that are active in the thylakoid membrane of the cyanobacterium.
Transposition of IS
1397
in the Family
Enterobacteriaceae
and First Characterization of ISKpn
1
, a New Insertion Sequence Associated with
Klebsiella pneumoniae
Palindromic Units
Authors: Wilde Caroline , Bachellier Sophie , Hofnung Maurice , Clément Jean-Marie 
Published: July 2002

ABSTRACT

IS
1397
and ISKpn
1
are IS
3
family members which are specifically inserted into the loop of palindromic units (PUs). IS
1397
is shown to transpose into PUs with sequences close or identical to the
Escherichia coli
consensus, even in other enterobacteria (
Salmonella enterica
serovar Typhimurium,
Klebsiella pneumoniae
, and
Klebsiella oxytoca
). Moreover, we show that homologous intergenic regions containing PUs constitute IS
1397
transpositional hot spots, despite bacterial interspersed mosaic element structures that differ among the three species. ISKpn
1
, described here for the first time, is specific for PUs from
K. pneumoniae
, in which we discovered it. A sequence comparison between the two insertion sequences allowed us to define a motif possibly accounting for their specificity.
Reconstruction of
Escherichia coli mrcA
(PBP 1a) Mutants Lacking Multiple Combinations of Penicillin Binding Proteins
Authors: Meberg Bernadette M. , Sailer Frances C. , Nelson David E. , Young Kevin D. 
Published: July 2002

ABSTRACT

Previously, we constructed a set of mutants from which eight penicillin binding protein (PBP) genes were deleted in 192 combinations from
Escherichia coli
(S. A. Denome, P. K. Elf, T. A. Henderson, D. E. Nelson, and K. D. Young, J. Bacteriol. 181:3981-3993, 1999). Although these mutants were constructed correctly as determined by restriction mapping and the absence of relevant protein products, we recently discovered by PCR mapping that strains from which
mrcA
(PBP 1a) was deleted were also missing two neighboring genes of unknown function (
yrfE
and
yrfF
). We created a new deletion mutation in
mrcA
and reconstructed 63 strains lacking PBP 1a and other PBP mutant combinations. The new
mrcA
mutants do not exhibit mucoidy, phage resistance, temperature sensitivity, growth rate defects, or antibiotic resistance, suggesting that these phenotypes require the loss of either
yrfE
or
yrfF
alone or in combination with the absence of multiple PBPs.
Integron Integrases Possess a Unique Additional Domain Necessary for Activity
Authors: Messier Nancy , Roy Paul H. 
Published: July 2002

ABSTRACT
Integrons are genetic elements capable of integrating genes by a site-specific recombination system catalyzed by an integrase. Integron integrases are members of the tyrosine recombinase family and possess the four invariant residues (RHRY) and conserved motifs (boxes I and II and patches I, II, and III). An alignment of integron integrases compared to other tyrosine recombinases shows an additional group of residues around the patch III motif. We have analyzed the DNA binding and recombination properties of class I integron integrase (IntI1) variants carrying mutations at residues that are well conserved among all tyrosine recombinases and at some residues from the additional motif that are conserved among the integron integrases. The well-conserved residues studied were H277 from the conserved tetrad RHRY (about 90% conserved), E121 found in the patch I motif (about 80% conserved in prokaryotic recombinases), K171 from the patch II motif (near 100% conserved), W229 and F233 from the patch III motif, and G302 of box II (about 80% conserved in prokaryotic recombinases). Additional IntI1 mutated residues were K219 and a deletion of the sequence ALER215. We observed that E121, K171, and G302 play a role in the recombination activity but can be mutated without disturbing binding to DNA. W229, F233, and the conserved histidine (H277) may be implicated in protein folding or DNA binding. Some of the extra residues of IntI1 seem to play a role in DNA binding (K219) while others are implicated in the recombination activity (ALER215 deletion).
RNA Expression Analysis Using an Antisense
Bacillus subtilis
Genome Array
Authors: Lee Jian-Ming , Zhang Shehui , Saha Soumitra , Santa Anna Sonia , Jiang Can , Perkins John 
Published: July 2002

ABSTRACT

We have developed an antisense oligonucleotide microarray for the study of gene expression and regulation in
Bacillus subtilis
by using Affymetrix technology. Quality control tests of the
B. subtilis
GeneChip were performed to ascertain the quality of the array. These tests included optimization of the labeling and hybridization conditions, determination of the linear dynamic range of gene expression levels, and assessment of differential gene expression patterns of known vitamin biosynthetic genes. In minimal medium, we detected transcripts for approximately 70% of the known open reading frames (ORFs). In addition, we were able to monitor the transcript level of known biosynthetic genes regulated by riboflavin, biotin, or thiamine. Moreover, novel transcripts were also detected within intergenic regions and on the opposite coding strand of known ORFs. Several of these novel transcripts were subsequently correlated to new coding regions.
Demonstration that the Group II Intron from the Clostridial Conjugative Transposon Tn
5397
Undergoes Splicing In Vivo
Authors: Roberts Adam P. , Braun Veit , von Eichel-Streiber Christoph , Mullany Peter 
Published: July 2002

ABSTRACT

Previous work has identified the conjugative transposon Tn
5397
from
Clostridium difficile
. This element was shown to contain a group II intron. Tn
5397
can be conjugatively transferred from
C. difficile
to
Bacillus subtilis
. In this work we show that the intron is spliced in both these hosts and that nonspliced RNA is also present. We constructed a mutation in the open reading frame within the intron, and this prevented splicing but did not prevent the formation of the circular form of the conjugative transposon (the likely transposition intermediate) or decrease the frequency of intergeneric transfer of Tn
5397
. Therefore, the intron is spliced, but splicing is not required for conjugation of Tn
5397
.
Genetic Footprinting in Bacteria
Authors: Hare Roberta S. , Walker Scott S. , Dorman Thomas E. , Greene Jonathan R. , Guzman Luz-Maria , Kenney Teresa J. , Sulavik Mark C. , Baradaran Khandan , Houseweart Chad , Yu Haiying , Foldes Zuzana , Motzer Anna , Walbridge Michael , Shimer George H. , Shaw Karen Joy 
Published: July 2002

ABSTRACT

In vivo genetic footprinting was developed in the yeast
Saccharomyces cerevisiae
to simultaneously assess the importance of thousands of genes for the fitness of the cell under any growth condition. We have developed in vivo genetic footprinting for
Escherichia coli
, a model bacterium and pathogen. We further demonstrate the utility of this technology for rapidly discovering genes that affect the fitness of
E. coli
under a variety of growth conditions. The definitive features of this system include a conditionally regulated Tn
10
transposase with relaxed sequence specificity and a conditionally regulated replicon for the vector containing the transposase and mini-Tn
10
transposon with an outwardly oriented promoter. This system results in a high frequency of randomly distributed transposon insertions, eliminating the need for the selection of a population containing transposon insertions, stringent suppression of transposon mutagenesis, and few polar effects. Successful footprints have been achieved for most genes longer than 400 bp, including genes located in operons. In addition, the ability of recombinant proteins to complement mutagenized hosts has been evaluated by genetic footprinting using a bacteriophage λ transposon delivery system.
Occurrence of Transsulfuration in Synthesis of
l
-Homocysteine in an Extremely Thermophilic Bacterium,
Thermus thermophilus
HB8
Authors: Yamagata Shuzo , Ichioka Kazuhito , Goto Koji , Mizuno Yasuko , Iwama Tomonori 
Published: July 2002

ABSTRACT

A cell extract of an extremely thermophilic bacterium,
Thermus thermophilus
HB8, cultured in a synthetic medium catalyzed cystathionine γ-synthesis with
O
-acetyl-
l
-homoserine and
l
-cysteine as substrates but not β-synthesis with
dl
-homocysteine and
l
-serine (or
O
-acetyl-
l
-serine). The amounts of synthesized enzymes metabolizing sulfur-containing amino acids were estimated by determining their catalytic activities in cell extracts. The syntheses of cysthathionine β-lyase (EC
4.4.1.8
) and
O
-acetyl-
l
-serine sulfhydrylase (EC
4.2.99.8
) were markedly repressed by
l
-methionine supplemented to the medium.
l
-Cysteine and glutathione, both at 0.5 mM, added to the medium as the sole sulfur source repressed the synthesis of
O
-acetylserine sulfhydrylase by 55 and 73%, respectively, confirming that this enzyme functions as a cysteine synthase. Methionine employed at 1 to 5 mM in the same way derepressed the synthesis of
O
-acetylserine sulfhydrylase 2.1- to 2.5-fold. A method for assaying a low concentration of sulfide (0.01 to 0.05 mM) liberated from homocysteine by determining cysteine synthesized with it in the presence of excess amounts of
O
-acetylserine and a purified preparation of the sulfhydrylase was established. The extract of cells catalyzed the homocysteine γ-lyase reaction, with a specific activity of 5 to 7 nmol/min/mg of protein, but not the methionine γ-lyase reaction. These results suggested that cysteine was also synthesized under the conditions employed by the catalysis of
O
-acetylserine sulfhydrylase using sulfur of homocysteine derived from methionine. Methionine inhibited
O
-acetylserine sulfhydrylase markedly. The effects of sulfur sources added to the medium on the synthesis of
O
-acetylhomoserine sulfhydrylase and the inhibition of the enzyme activity by methionine were mostly understood by assuming that the organism has two proteins having
O
-acetylhomoserine sulfhydrylase activity, one of which is cystathionine γ-synthase. Although it has been reported that homocysteine is directly synthesized in
T. thermophilus
HB27 by the catalysis of
O
-acetylhomoserine sulfhydrylase on the basis of genetic studies (T. Kosuge, D. Gao, and T. Hoshino, J. Biosci. Bioeng. 90:271–279, 2000), the results obtained in this study for the behaviors of related enzymes indicate that sulfur is first incorporated into cysteine and then transferred to homocysteine via cystathionine in
T. thermophilus
HB8.
Characterization of
Brucella suis clpB
and
clpAB
Mutants and Participation of the Genes in Stress Responses
Authors: Ekaza Euloge , Teyssier Jacques , Ouahrani-Bettache Safia , Liautard Jean-Pierre , Köhler Stephan 
Published: July 2002

ABSTRACT

Pathogens often encounter stressful conditions inside their hosts. In the attempt to characterize the stress response in
Brucella suis
, a gene highly homologous to
Escherichia coli clpB
was isolated from
Brucella suis
, and the deduced amino acid sequence showed features typical of the ClpB ATPase family of stress response proteins. Under high-temperature stress conditions, ClpB of
B. suis
was induced, and an isogenic
B. suis clpB
mutant showed increased sensitivity to high temperature, but also to ethanol stress and acid pH. The effects were reversible by complementation. Simultaneous inactivation of
clpA
and
clpB
resulted in a mutant that was sensitive to oxidative stress. In
B. suis
expressing
gfp
, ClpA but not ClpB participated in degradation of the green fluorescent protein at 42°C. We concluded that ClpB was responsible for tolerance to several stresses and that the lethality caused by harsh environmental conditions may have similar molecular origins.
Identification of a Lycopene β-Cyclase Required for Bacteriorhodopsin Biogenesis in the ArchaeonHalobacterium salinarum
Authors: Peck Ronald F. , Johnson Eric A. , Krebs Mark P. 
Published: July 2002

ABSTRACTBiogenesis of the light-driven proton pump bacteriorhodopsin in the archaeonHalobacterium salinarumrequires coordinate synthesis of the bacterioopsin apoprotein and carotenoid precursors of retinal, which serves as a covalently bound cofactor. As a step towards elucidating the mechanism and regulation of carotenoid metabolism during bacteriorhodopsin biogenesis, we have identified anH. salinarumgene required for conversion of lycopene to β-carotene, a retinal precursor. The gene, designatedcrtY, is predicted to encode an integral membrane protein homologous to lycopene β-cyclases identified in bacteria and fungi. To testcrtYfunction, we constructedH. salinarumstrains with in-frame deletions in the gene. In the deletion strains, bacteriorhodopsin, retinal, and β-carotene were undetectable, whereas lycopene accumulated to high levels (≈1.3 nmol/mg of total cell protein). Heterologous expression ofH. salinarum crtYin a lycopene-producingEscherichia colistrain resulted in β-carotene production. These results indicate thatH. salinarum crtYencodes a functional lycopene β-cyclase required for bacteriorhodopsin biogenesis. Comparative sequence analysis yields a topological model of the protein and provides a plausible evolutionary connection between heterodimeric lycopene cyclases in bacteria and bifunctional lycopene cyclase-phytoene synthases in fungi.