bluuebunny/crossref_metadata_2025_split huggingface dataset:
GIL16, a New Gram-Positive Tectiviral Phage Related to theBacillus thuringiensisGIL01 and theBacillus cereuspBClin15 Elements
Authors: Verheust Céline , Fornelos Nadine , Mahillon Jacques 
Published: March 2005

ABSTRACTOne of the most notable characteristics ofTectiviridaeresides in their double-layer coats: the double-stranded DNA is located within a flexible lipoprotein vesicle covered by a rigid protein capsid. Despite their apparent rarity, tectiviruses have an extremely wide distribution compared to other phage groups. Members of this family have been found to infect gram-negative (PRD1 and relatives) as well as gram-positive (Bam35, GIL01, AP50, and φNS11) hosts. Several reports have shown that tectiviruses infecting gram-negative bacteria are closely related, whereas no information is currently available on the genetic relationship among those infecting gram-positive bacteria. The present study reports the sequence of GIL16, a new isolate originating fromBacillus thuringiensis, and a genetic comparison of this isolate with the tectiviral bacteriophages Bam35 and GIL01, which originated fromB. thuringiensisserovars Alesti and Israelensis, respectively. In contrast to PRD1 and its relatives, these are temperate bacteriophages existing as autonomous linear prophages within the host cell. Mutations in a particular motif in both the GIL01 and GIL16 phages are also shown to correlate with a switch to the lytic cycle. Interestingly, both bacterial viruses displayed narrow, yet slightly different, host spectrums. We also explore the hypothesis that pBClin15, a linear plasmid hosted by theBacillus cereusreference strain ATCC 14579, is also a prophage. Sequencing of its inverted repeats at both extremities and a comparison with GIL01 and GIL16 emphasize its relationship to theTectiviridae.
In Vivo Evidence for a
recA
-Independent Recombination Process in
Escherichia coli
That Permits Completion of Replication of DNA Containing UV Damage in Both Strands
Authors: Ozgenc Ali I. , Szekeres Edward S. , Lawrence Christopher W. 
Published: March 2005

ABSTRACT

We have investigated recombination mechanisms promoting the completion of replication in the face of unrepaired DNA damage by transforming an isogenic set of
uvrA6
excision-defective
Escherichia coli
strains with pUC-based plasmids in which each strand carried, at staggered positions, a single thymine-thymine pyrimidine (6-4) pyrimidinone lesion. The distance between the lesions was 28 or 8 bp in one orientation relative to the unidirectional ColE1 origin of replication or, in the other orientation, 30 or 10 bp. C-C mismatches placed opposite each of the T-T photoproducts permit unambiguous detection of the three events that can lead to the completion of replication: sister-strand recombination, translesion replication (TR) on the leading strand, and TR on the lagging strand. We find that
E. coli
possesses a largely constitutive,
recA
-independent sister-strand recombination mechanism that allows 9% or more of these severely compromised plasmids to be fully replicated. In one orientation, such recombination depends partly on
recG
and
priA
but not on
ruvA
,
ruvB
,
ruvC
, or
mutS
and is largely independent of
recF
. In the other orientation, recombination is dependent on none of the genes. The strains used did not contain the cryptic phage encoding
recET
, which encodes enzymes that promote interplasmid recombination. The nature of the
recA
-independent recombination mechanism is not known but could perhaps result from a template-strand-switching, or copy choice, process.
HetR-Dependent and -Independent Expression of Heterocyst-Related Genes in an
Anabaena
Strain Overproducing the NtcA Transcription Factor
Authors: Olmedo-Verd Elvira , Flores Enrique , Herrero Antonia , Muro-Pastor Alicia M. 
Published: March 2005

ABSTRACT

Heterocyst development in the cyanobacterium
Anabaena
sp. strain PCC 7120 depends on both the global nitrogen control transcription factor NtcA and the cell differentiation regulatory protein HetR, with expression of nt
cA
and
hetR
being dependent on each other. In this study we constructed strains that constitutively express the nt
cA
gene leading to high levels of NtcA protein irrespective of the nitrogen source, and we analyzed the effects of such NtcA levels on heterocyst differentiation. In the NtcA-overproducing strain, heterocyst differentiation, induction of NtcA-dependent heterocyst development genes or operons such as
devBCA
or the
cox2
operon, and NtcA-dependent excision of the 11-kb
nifD
-intervening element only took place under nitrogen deficiency. Although functional heterocysts were produced in response to nitrogen step-down, the NtcA overproducing strain could not grow diazotrophically. Overexpression of
ntcA
in a
hetR
background promoted expression of
devBCA
in response to ammonium withdrawal and excision of the 11-kb element even in the presence of combined nitrogen. Our results show that some NtcA-dependent heterocyst-related genes can be expressed independently of HetR.
Functional Domains of NosR, a Novel Transmembrane Iron-Sulfur Flavoprotein Necessary for Nitrous Oxide Respiration
Authors: Wunsch Patrick , Zumft Walter G. 
Published: March 2005

ABSTRACT

Bacterial nitrous oxide (N
2
O) respiration depends on the polytopic membrane protein NosR for the expression of N
2
O reductase from the
nosZ
gene. We constructed His-tagged NosR and purified it from detergent-solubilized membranes of
Pseudomonas stutzeri
ATCC 14405. NosR is an iron-sulfur flavoprotein with redox centers positioned at opposite sides of the cytoplasmic membrane. The flavin cofactor is presumably bound covalently to an invariant threonine residue of the periplasmic domain. NosR also features conserved CX
3
CP motifs, located C-terminally of the transmembrane helices TM4 and TM6. We genetically manipulated
nosR
with respect to these different domains and putative functional centers and expressed recombinant derivatives in a
nosR
null mutant, MK418
nosR
::Tn
5
. NosR's function was studied by its effects on N
2
O respiration, NosZ synthesis, and the properties of purified NosZ proteins. Although all recombinant NosR proteins allowed the synthesis of NosZ, a loss of N
2
O respiration was observed upon deletion of most of the periplasmic domain or of the C-terminal parts beyond TM2 or upon modification of the cysteine residues in a highly conserved motif, CGWLCP, following TM4. Nonetheless, NosZ purified from the recombinant NosR background exhibited in vitro catalytic activity. Certain NosR derivatives caused an increase in NosZ of the spectral contribution from a modified catalytic Cu site. In addition to its role in
nosZ
expression, NosR supports in vivo N
2
O respiration. We also discuss its putative functions in electron donation and redox activation.
Substrate Specificities and Expression Patterns Reflect the Evolutionary Divergence of Maltose ABC Transporters in
Thermotoga maritima
Authors: Nanavati Dhaval M. , Nguyen Tu N. , Noll Kenneth M. 
Published: March 2005

ABSTRACT

Duplication of transporter genes is apparent in the genome sequence of the hyperthermophilic bacterium
Thermotoga maritima
. The physiological impacts of these duplications are not well understood, so we used the bacterium's two putative maltose transporters to begin a study of the evolutionary relationship between a transporter's function and the control of expression of its genes. We show that the substrate binding proteins encoded by these operons, MalE1 and MalE2, have different substrate specificities and affinities and that they are expressed under different growth conditions. MalE1 binds maltose (dissociation constant [

K
D

], 24 ± 1 μM), maltotriose (

K
D

, 8 ± 0.5 nM), and β-(1→4)-mannotetraose (

K
D

, 38 ± 1 μM). In contrast, MalE2 binds maltose (

K
D

, 8.4 ± 1 μM), maltotriose (

K
D

, 11.5 ± 1.5 μM), and trehalose (

K
D

, 9.5 ± 1.0 μM) confirming the findings of Wassenberg et al. (J. Mol. Biol. 295:279-288, 2000). Neither protein binds lactose. We examined the expression of these operons at both the transcriptional and translational levels and found that MalE1 is expressed in cells grown on lactose or guar gum and that MalE2 is highly expressed in starch- and trehalose-grown cells. Evidence is provided that
malE1
,
malF1
, and perhaps
malG1
are cotranscribed and so constitute an operon. An open reading frame encoding a putative transcriptional regulatory protein adjacent to this operon (TM1200) is also up-regulated in response to growth on lactose. These evolutionarily related transporter operons have diverged both in function and expression to assume apparently different physiological roles.
The Rok Protein of
Bacillus subtilis
Represses Genes for Cell Surface and Extracellular Functions
Authors: Albano Mark , Smits Wiep Klaas , Ho Linh T. Y. , Kraigher Barbara , Mandic-Mulec Ines , Kuipers Oscar P. , Dubnau David 
Published: March 2005

ABSTRACT

Rok is a repressor of the transcriptional activator ComK and is therefore an important regulator of competence in
Bacillus subtilis
(T. T. Hoa, P. Tortosa, M. Albano, and D. Dubnau, Mol. Microbiol. 43:15-26, 2002). To address the wider role of Rok in the physiology of
B. subtilis
, we have used a combination of transcriptional profiling, gel shift experiments, and the analysis of
lacZ
fusions. We demonstrate that Rok is a repressor of a family of genes that specify membrane-localized and secreted proteins, including a number of genes that encode products with antibiotic activity. We present evidence for the recent introduction of
rok
into the
B. subtilis-Bacillus licheniformis-Bacilllus amyloliquefaciens
group by horizontal transmission.
Cytochrome
bd
Oxidase, Oxidative Stress, and Dioxygen Tolerance of the Strictly Anaerobic Bacterium
Moorella thermoacetica
Authors: Das Amaresh , Silaghi-Dumitrescu Radu , Ljungdahl Lars G. , Kurtz Donald M. 
Published: March 2005

ABSTRACT

The gram-positive, thermophilic, acetogenic bacterium
Moorella thermoacetica
can reduce CO
2
to acetate via the Wood-Ljungdahl (acetyl coenzyme A synthesis) pathway. This report demonstrates that, despite its classification as a strict anaerobe,
M. thermoacetica
contains a membrane-bound cytochrome
bd
oxidase that can catalyze reduction of low levels of dioxygen. Whole-cell suspensions of
M. thermoacetica
had significant endogenous O
2
uptake activity, and this activity was increased in the presence of methanol or CO, which are substrates in the Wood-Ljungdahl pathway. Cyanide and azide strongly (∼70%) inhibited both the endogenous and CO/methanol-dependent O
2
uptake. UV-visible light absorption and electron paramagnetic resonance spectra of
n
-dodecyl-β-maltoside extracts of
M. thermoacetica
membranes showed the presence of a cytochrome
bd
oxidase complex containing cytochrome
b
561
, cytochrome
b
595
, and cytochrome
d
(chlorin). Subunits I and II of the
bd
oxidase were identified by N-terminal amino acid sequencing. The
M. thermoacetica
cytochrome
bd
oxidase exhibited cyanide-sensitive quinol oxidase activity. The
M. thermoacetica
cytochrome
bd
(
cyd
) operon consists of four genes, encoding subunits I and II along with two ABC-type transporter proteins, homologs of which in other bacteria are required for assembly of the
bd
complex. The level of this
cyd
operon transcript was significantly increased when
M. thermoacetica
was grown in the absence of added reducing agent (cysteine + H
2
S). Expression of a 35-kDa cytosolic protein, identified as a cysteine synthase (CysK), was also induced by the nonreducing growth conditions. The combined evidence indicates that cytochrome
bd
oxidase and cysteine synthase protect against oxidative stress and contribute to the limited dioxygen tolerance of
M. thermoacetica
.
A Tetrahydrofolate-Dependent
O
-Demethylase, LigM, Is Crucial for Catabolism of Vanillate and Syringate in
Sphingomonas paucimobilis
SYK-6
Authors: Abe Tomokuni , Masai Eiji , Miyauchi Keisuke , Katayama Yoshihiro , Fukuda Masao 
Published: March 2005

ABSTRACT

Vanillate and syringate are converted into protocatechuate (PCA) and 3-
O
-methylgallate (3MGA), respectively, by
O
-demethylases in
Sphingomonas paucimobilis
SYK-6. PCA is further degraded via the PCA 4,5-cleavage pathway, while 3MGA is degraded through multiple pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and an unidentified 3MGA
O
-demethylase and gallate dioxygenase are participants. For this study, we isolated a 4.7-kb SmaI fragment that conferred on
Escherichia coli
the activity required for the conversion of vanillate to PCA. The nucleotide sequence of this fragment revealed an open reading frame of 1,413 bp (
ligM
), the deduced amino acid sequence of which showed 49% identity with that of the tetrahydrofolate (H
4
folate)-dependent syringate
O
-demethylase gene (
desA
). The
metF
and
ligH
genes, which are thought to be involved in H
4
folate-mediated C
1
metabolism, were located just downstream of
ligM
. The crude LigM enzyme expressed in
E. coli
converted vanillate and 3MGA to PCA and gallate, respectively, with similar specific activities, and only in the presence of H
4
folate; however, syringate was not a substrate for LigM. The disruption of
ligM
led to significant growth retardation on both vanillate and syringate, indicating that
ligM
is involved in the catabolism of these substrates. The ability of the
ligM
mutant to transform vanillate was markedly decreased, and this mutant completely lost the 3MGA
O
-demethylase activity. A
ligM desA
double mutant completely lost the ability to transform vanillate, thus indicating that
desA
also contributes to vanillate degradation. All of these results indicate that
ligM
encodes vanillate/3MGA
O
-demethylase and plays an important role in the O demethylation of vanillate and 3MGA, respectively.
CpxR/OmpR Interplay Regulates Curli Gene Expression in Response to Osmolarity inEscherichia coli
Authors: Jubelin Gregory , Vianney Anne , Beloin Christophe , Ghigo Jean-Marc , Lazzaroni Jean-Claude , Lejeune Philippe , Dorel Corinne 
Published: March 2005

ABSTRACTCurli fibers could be described as a virulence factor able to confer adherence properties to both abiotic and eukaryotic surfaces. The ability to adapt rapidly to changing environmental conditions through signal transduction pathways is crucial for the growth and pathogenicity of bacteria. OmpR was shown to activatecsgDexpression, resulting in curli production. The CpxR regulator was shown to negatively affect curli gene expression when binding to its recognition site that overlaps thecsgDOmpR-binding site. This study was undertaken to clarify how the interplay between the two regulatory proteins, OmpR and CpxR, can affect the transcription of the curli gene in response to variation of the medium osmolarity. Band-shift assays with purified CpxR proteins indicate that CpxR binds to thecsgDpromoter region at multiple sites that are ideally positioned to explain thecsgrepression activity of CpxR. To understand the physiological meaning of this in vitro molecular phenomenon, we analyzed the effects of an osmolarity shift on the two-component pathway CpxA/CpxR. We establish here that the Cpx pathway is activated at both transcriptional and posttranscriptional levels in response to a high osmolarity medium and that CpxR repressescsgDexpression in high-salt-content medium, resulting in low curli production. However,csgDrepression in response to high sucrose content is not mediated by CpxR but by the global regulatory protein H-NS. Therefore, multiple systems (EnvZ/OmpR, Cpx, Rcs, and H-NS) appear to be involved in sensing environmental osmolarity, leading to sophisticated regulation of the curli genes.
Nucleotide Sequence of the Head Assembly Gene Cluster of Bacteriophage L and Decoration Protein Characterization
Authors: Gilcrease Eddie B. , Winn-Stapley Danella A. , Hewitt F. Curtis , Joss Lisa , Casjens Sherwood R. 
Published: March 2005

ABSTRACT

The temperate
Salmonella enterica
bacteriophage L is a close relative of the very well studied bacteriophage P22. In this study we show that the L procapsid assembly and DNA packaging genes, which encode terminase, portal, scaffold, and coat proteins, are extremely close relatives of the homologous P22 genes (96.3 to 99.1% identity in encoded amino acid sequence). However, we also identify an L gene,
dec
, which is not present in the P22 genome and which encodes a protein (Dec) that is present on the surface of L virions in about 150 to 180 molecules/virion. We also show that the Dec protein is a trimer in solution and that it binds to P22 virions in numbers similar to those for L virions. Its binding dramatically stabilizes P22 virions against disruption by a magnesium ion chelating agent. Dec protein binds to P22 coat protein shells that have expanded naturally in vivo or by sodium dodecyl sulfate treatment in vitro but does not bind to unexpanded procapsid shells. Finally, analysis of phage L restriction site locations and a number of patches of nucleotide sequence suggest that phages ST64T and L are extremely close relatives, perhaps the two closest relatives that have been independently isolated to date among the lambdoid phages.
Magnesium Promotes Flagellation ofVibrio fischeri
Authors: O'Shea Therese M. , DeLoney-Marino Cindy R. , Shibata Satoshi , Aizawa Shin-Ichi , Wolfe Alan J. , Visick Karen L. 
Published: March 2005

ABSTRACTThe bacteriumVibrio fischerirequires bacterial motility to initiate colonization of the Hawaiian squidEuprymna scolopes. Once colonized, however, the bacterial population becomes largely unflagellated. To understand environmental influences onV. fischerimotility, we investigated migration of this organism in tryptone-based soft agar media supplemented with different salts. We found that optimal migration required divalent cations and, in particular, Mg2+. At concentrations naturally present in seawater, Mg2+improved migration without altering the growth rate of the cells. Transmission electron microscopy and Western blot experiments suggested that Mg2+addition enhanced flagellation, at least in part through an effect on the steady-state levels of flagellin protein.
Cyclic AMP (cAMP) and cAMP Receptor Protein Influence both Synthesis and Uptake of Extracellular Autoinducer 2 inEscherichia coli
Authors: Wang Liang , Hashimoto Yoshifumi , Tsao Chen-Yu , Valdes James J. , Bentley William E. 
Published: March 2005

ABSTRACTBacterial autoinducer 2 (AI-2) is proposed to be an interspecies mediator of cell-cell communication that enables cells to operate at the multicellular level. Many environmental stimuli have been shown to affect the extracellular AI-2 levels, carbon sources being among the most important. In this report, we show that both AI-2 synthesis and uptake inEscherichia coliare subject to catabolite repression through the cyclic AMP (cAMP)-CRP complex, which directly stimulates transcription of thelsr(for “luxSregulated”) operon and indirectly repressesluxSexpression. Specifically, cAMP-CRP is shown to bind to a CRP binding site located in the upstream region of thelsrpromoter and works with the LsrR repressor to regulate AI-2 uptake. The functions of thelsroperon and its regulators, LsrR and LsrK, previously reported inSalmonella entericaserovar Typhimurium, are confirmed here forE. coli.The elucidation of cAMP-CRP involvement inE. coliautoinduction impacts many areas, including the growth ofE. coliin fermentation processes.
Characterization of a Novel Zinc-Containing, Lysine-Specific Aminopeptidase from the Hyperthermophilic ArchaeonPyrococcus furiosus
Authors: Story Sherry V. , Shah Claudia , Jenney Francis E. , Adams Michael W. W. 
Published: March 2005

ABSTRACTCell extracts of the proteolytic, hyperthermophilic archaeonPyrococcus furiosuscontain high specific activity (11 U/mg) of lysine aminopeptidase (KAP), as measured by the hydrolysis ofl-lysyl-p-nitroanilide (Lys-pNA). The enzyme was purified by multistep chromatography. KAP is a homotetramer (38.2 kDa per subunit) and, as purified, contains 2.0 ± 0.48 zinc atoms per subunit. Surprisingly, its activity was stimulated fourfold by the addition of Co2+ions (0.2 mM). Optimal KAP activity with Lys-pNA as the substrate occurred at pH 8.0 and a temperature of 100°C. The enzyme had a narrow substrate specificity with di-, tri-, and tetrapeptides, and it hydrolyzed only basic N-terminal residues at high rates. Mass spectroscopy analysis of the purified enzyme was used to identify, in theP. furiosusgenome database, a gene (PF1861) that encodes a product corresponding to 346 amino acids. The recombinant protein containing a polyhistidine tag at the N terminus was produced inEscherichia coliand purified using affinity chromatography. Its properties, including molecular mass, metal ion dependence, and pH and temperature optima for catalysis, were indistinguishable from those of the native form, although the thermostability of the recombinant form was dramatically lower than that of the native enzyme (half-life of approximately 6 h at 100°C). Based on its amino acid sequence, KAP is part of the M18 family of peptidases and represents the first prokaryotic member of this family. KAP is also the first lysine-specific aminopeptidase to be purified from an archaeon.
Molecular Analysis of the Rebeccamycin
l
-Amino Acid Oxidase from
Lechevalieria aerocolonigenes
ATCC 39243
Authors: Nishizawa Tomoyasu , Aldrich Courtney C. , Sherman David H. 
Published: March 2005

ABSTRACT

Rebeccamycin, a member of the tryptophan-derived indolocarbazole family, is produced by
Lechevalieria aerocolonigenes
ATCC 39243. The biosynthetic pathway that specifies biosynthesis of this important metabolite is comprised of 11 genes spanning 18 kb of DNA. A presumed early enzyme involved in elaboration of the rebeccamycin aglycone is encoded by
rebO
, located at the left-hand region of the
reb
gene cluster. The deduced protein product, RebO (51.9 kDa), is an
l
-amino acid oxidase (
l
-AAO) that has 27% identity to an
l
-AAO from
Scomber japonicus
(animal, mackerel) and is a member of the family of FAD-dependent oxidase enzymes. In order to study the biochemical properties of this key enzyme, the
rebO
gene was overexpressed and purified from
Escherichia coli
. Biochemical characterization showed that RebO is dimeric, with a molecular mass of approximately 101 kDa. Further analysis revealed that the enzyme contains a noncovalently bound FAD cofactor and is reoxidized at the expense of molecular oxygen by producing one molecule of hydrogen peroxide. Based on kinetic studies, RebO shows significant preference for 7-chloro-
l
-tryptophan, suggesting its likely role as the natural early pathway substrate. Furthermore, the native RebO enzyme has evident, albeit limited, flexibility as shown by bioconversion studies with unnatural substrates. This work provides the first analysis of a structural enzyme involved in construction of this important class of indolocarbazole natural products.