bluuebunny/crossref_metadata_2025_split huggingface dataset:
Antisense RNA Regulation by Stable Complex Formation in theEnterococcus faecalisPlasmid pAD1parAddiction System
Authors: Weaver Keith E. , Ehli Erik A. , Nelson Jessica S. , Patel Smita 
Published: September 2004

ABSTRACTTheparstability determinant, encoded by theEnterococcus faecalisplasmid pAD1, is the only antisense RNA regulated postsegregational killing system identified in gram-positive bacteria. Because of the unique organization of theparlocus, theparantisense RNA, RNA II, binds to its target, RNA I, at relatively small, interspersed regions of complementarity. The results of this study suggest that, rather than targeting the antisense bound message for rapid degradation, as occurs in most other antisense RNA regulated systems, RNA I and RNA II form a relatively stable, presumably translationally inactive complex. The stability of the RNA I-RNA II complex would allow RNA I to persist in an untranslated state unless or until the encoding plasmid was lost. After plasmid loss, RNA II would be removed from the complex, allowing translational activation of RNA I. The mechanism of RNA I activation in vivo is unknown, but in vitro dissociation experiments suggest that active removal of RNA II, for example by a cellular RNase, may be required.
Characterization of Three New Competence-Regulated Operons in
Haemophilus influenzae
Authors: VanWagoner Timothy M. , Whitby Paul W. , Morton Daniel J. , Seale Thomas W. , Stull Terrence L. 
Published: September 2004

ABSTRACT

Haemophilus influenzae
is one of a growing number of bacteria in which the natural ability to uptake exogenous DNA for potential genomic transformation has been recognized. To date, several operons involved in transformation in this organism have been described. These operons are characterized by a conserved 22-bp regulatory element upstream of the first gene and are induced coincident with transfer from rich to nutrient-depleted media. The previously identified operons comprised genes encoding proteins that include members of the type II secretion system and type IV pili, shown to be essential for transformation in other bacteria, and other proteins previously identified as required for transformation in
H. influenzae
. In the present study, three novel competence operons were identified by comparative genomics and transcriptional analysis. These operons have been further characterized by construction of null mutants and examination of the resulting transformation phenotypes. The putative protein encoded by the HI0366 gene was shown to be essential for DNA uptake, but not binding, and is homologous to a protein shown to be required for pilus biogenesis and twitching motility in
Pseudomonas aeruginosa
. An insertion in HI0939 abolished both DNA binding and uptake. The predicted product of this gene shares characteristics with PulJ, a pseudopilin involved in pullulanase export in
Klebsiella oxytoca
.
A
Francisella tularensis
Pathogenicity Island Required for Intramacrophage Growth
Authors: Nano Francis E. , Zhang Na , Cowley Siobhán C. , Klose Karl E. , Cheung Karen K. M. , Roberts Michael J. , Ludu Jagjit S. , Letendre Gregg W. , Meierovics Anda I. , Stephens Gwen , Elkins Karen L. 
Published: September 2004

ABSTRACT

Francisella tularensis
is a gram-negative, facultative intracellular pathogen that causes the highly infectious zoonotic disease tularemia. We have discovered a ca. 30-kb pathogenicity island of
F. tularensis
(FPI) that includes four large open reading frames (ORFs) of 2.5 to 3.9 kb and 13 ORFs of 1.5 kb or smaller. Previously, two small genes located near the center of the FPI were shown to be needed for intramacrophage growth. In this work we show that two of the large ORFs, located toward the ends of the FPI, are needed for virulence. Although most genes in the FPI encode proteins with amino acid sequences that are highly conserved between high- and low-virulence strains, one of the FPI genes is present in highly virulent type A
F. tularensis
, absent in moderately virulent type B
F. tularensis
, and altered in
F. tularensis
subsp.
novicida
, which is highly virulent for mice but avirulent for humans. The G+C content of a 17.7-kb stretch of the FPI is 26.6%, which is 6.6% below the average G+C content of the
F. tularensis
genome. This extremely low G+C content suggests that the DNA was imported from a microbe with a very low G+C-containing chromosome.
Genomic Changes Arising in Long-Term Stab Cultures ofEscherichia coli
Authors: Faure D. , Frederick R. , Włoch D. , Portier P. , Blot M. , Adams J. 
Published: September 2004

ABSTRACTGenomic scans of clones isolated from long-term stab cultures ofEscherichia coliK-12 showed the loss of two large segments of the genome, with each lost segment being approximately 20 kb long. A detailed analysis of one of the deletions, located between 5.4 and 5.9 min, revealed that similar deletions had arisen in several other stab cultures. All deletions of this type exhibited a right terminus ending precisely at an IS5Aelement and a left terminus that varied over an ∼5-kb range but was bordered in all but two cases by sequences belonging to the preferred consensus target sequence for IS5, YTAR. The ubiquity of such deletions in independent stab cultures and the increase in their frequency over time argue that they have a selective advantage. It is speculated that the loss of thecrllocus is responsible for the selective advantage of the deletions.
The Fur Homologue inBorrelia burgdorferi
Authors: Katona Laura I. , Tokarz Rafal , Kuhlow Christopher J. , Benach Jordi , Benach Jorge L. 
Published: September 2004

ABSTRACTBorrelia burgdorfericontains a gene that codes for a Fur homologue. The function of this Fur protein is unknown; however, spirochetes grown at 23 or 35°C expressedfuras determined by reverse transcriptase PCR. Thefurgene (BB0647) was cloned and overexpressed as a His-Fur fusion protein inEscherichia coli. The fusion protein was purified by zinc-chelate chromatography, and the N-terminal His tag was removed to generate recombinant Fur for use in mobility shift studies. Fur bound DNA containing theE. coliFur box sequence (GATAATGATAATCATTATC) orBacillus subtilisPer box sequence (TTATAAT-ATTATAA) with an apparentKdof ∼20 nM. Fur also bound the upstream sequences of threeBorreliagenes: BB0646 (gene encoding a hydrolase of the α/β-fold family), BB0647 (fur), and BB0690 (napA). Addition of metal ions was not required. Binding activity was greatly decreased by either exposure to oxidizing agents (H2O2,t-butyl hydroperoxide, cumene hydroperoxide, or diamide) or by addition of Zn2+.B. burgdorferiNapA is a homologue of Dps. Dps functions inE. colito protect DNA against damage during periods of redox stress. Fur may function inB. burgdorferias a repressor and regulate oxidative stress genes. Additional genes (10 chromosomal and 15 plasmid) that may be Fur regulated were identified by in silico analysis.
Hetero- and Autoprocessing of the Extracellular Metalloprotease (Mpr) in
Bacillus subtilis
Authors: Park Chi Hye , Lee Sang Jun , Lee Sung Gu , Lee Weon Sup , Byun Si Myung 
Published: September 2004

ABSTRACT

Most proteases are synthesized as inactive precursors which are processed by proteolytic cleavage into a mature active form, allowing regulation of their proteolytic activity. The activation of the glutamic-acid-specific extracellular metalloprotease (Mpr) of
Bacillus subtilis
has been examined. Analysis of Mpr processing in defined protease-deficient mutants by activity assay and Western blotting revealed that the extracellular protease Bpr is required for Mpr processing. pro-Mpr remained a precursor form in
bpr
-deficient strains, and glutamic-acid-specific proteolytic activity conferred by Mpr was not activated in
bpr
-deficient strains. Further, purified pro-Mpr was processed to an active form by purified Bpr protease in vitro. We conclude that Mpr is activated by Bpr in vivo, and that heteroprocessing, rather than autoprocessing, is the major mechanism of Mpr processing in vivo. Exchange of glutamic acid for serine in the cleavage site of Mpr (S93E) allowed processing of Mpr into its mature form, regardless of the presence of other extracellular proteases, including Bpr. Thus, a single amino acid change is sufficient to convert the Mpr processing mechanism from heteroprocessing to autoprocessing.
Bacillus subtilis
ResD Induces Expression of the Potential Regulatory Genes
yclJK
upon Oxygen Limitation
Authors: Härtig Elisabeth , Geng Hao , Hartmann Anja , Hubacek Angela , Münch Richard , Ye Rick W. , Jahn Dieter , Nakano Michiko M. 
Published: September 2004

ABSTRACT

Transcription of the
yclJK
operon, which encodes a potential two-component regulatory system, is activated in response to oxygen limitation in
Bacillus subtilis
. Northern blot analysis and assays of
yclJ-lacZ
reporter gene fusion activity revealed that the anaerobic induction is dependent on another two-component signal transduction system encoded by
resDE
. ResDE was previously shown to be required for the induction of anaerobic energy metabolism. Electrophoretic mobility shift assays and DNase I footprinting experiments showed that the response regulator ResD binds specifically to the
yclJK
regulatory region upstream of the transcriptional start site. In vitro transcription experiments demonstrated that ResD is sufficient to activate
yclJ
transcription. The phosphorylation of ResD by its sensor kinase, ResE, highly stimulates its activity as a transcriptional activator. Multiple nucleotide substitutions in the ResD binding regions of the
yclJ
promoter abolished ResD binding in vitro and prevented the anaerobic induction of
yclJK
in vivo. A weight matrix for the ResD binding site was defined by a bioinformatic approach. The results obtained suggest the existence of a new branch of the complex regulatory system employed for the adaptation of
B. subtilis
to anaerobic growth conditions.
Contribution of the Mismatch DNA Repair System to the Generation of Stationary-Phase-Induced Mutants of
Bacillus subtilis
Authors: Pedraza-Reyes Mario , Yasbin Ronald E. 
Published: September 2004

ABSTRACT

A reversion assay system previously implemented to demonstrate the existence of adaptive or stationary-phase-induced mutagenesis in
Bacillus subtilis
was utilized in this report to study the influence of the mismatch DNA repair (MMR) system on this type of mutagenesis. Results revealed that a strain deficient in MutSL showed a significant propensity to generate increased numbers of stationary-phase-induced revertants. These results suggest that absence or depression of MMR is an important factor in the mutagenesis of nongrowing
B. subtilis
cells because of the role of MMR in repairing DNA damage. In agreement with this suggestion, a significant decrease in the number of adaptive revertant colonies, for the three markers tested, occurred in
B. subtilis
cells which overexpressed a component of the MMR system. Interestingly, the single overexpression of
mutS
, but not of
mutL
, was sufficient to decrease the level of adaptive mutants in the reversion assay system of
B. subtilis
. The results presented in this work, as well as in our previous studies, appear to suggest that an MMR deficiency, putatively attributable to inactivation or saturation with DNA damage of MutS, may occur in a subset of
B. subtilis
cells that differentiate into the hypermutable state.
Two Proteins with Ornithine Acetyltransferase Activity Show Different Functions in
Streptomyces clavuligerus:
Oat2 Modulates Clavulanic Acid Biosynthesis in Response to Arginine
Authors: de la Fuente A. , Martín J. F. , Rodríguez-García A. , Liras P. 
Published: September 2004

ABSTRACT

The
oat2
gene, located in the clavulanic acid gene cluster in
Streptomyces clavuligerus
, is similar to
argJ
, which encodes
N
-acetylornithine:glutamic acid acetyltransferase activity. Purified proteins obtained by expression in
Escherichia coli
of the
argJ
and
oat2
genes of
S. clavuligerus
posses
N
-acetyltransferase activity. The kinetics and substrate specificities of both proteins are very similar. Deletion of the
oat2
gene did not affect the total
N
-acetylornithine transferase activity and slightly reduced the formation of clavulanic acid under standard culture conditions. However, the
oat2
mutant produced more clavulanic acid than the parental strain in cultures supplemented with high levels (above 1 mM) of arginine. The purified
S. clavuligerus
ArgR protein bound the arginine box in the
oat2
promoter, and the expression of
oat2
was higher in mutants with a disruption in
argR
(arginine-deregulated), confirming that the Arg boxes of
oat2
are functional in vivo. Our results suggest that the Oat2 protein or one of its reaction products has a regulatory role that modulates clavulanic acid biosynthesis in response to high arginine concentrations.
N-Linked Protein Glycosylation Is Required for Full Competence in
Campylobacter jejuni
81-176
Authors: Larsen Joseph C. , Szymanski Christine , Guerry Patricia 
Published: September 2004

ABSTRACT

The recent sequencing of the virulence plasmid of
Campylobacter jejuni
81-176 revealed the presence of genes homologous to type IV secretion systems (TFSS) that have subsequently been found in
Helicobacter pylori
and
Wolinella succinogenes
. Mutational analyses of some of these genes have implicated their involvement in intestinal epithelial cell invasion and natural competence. In this report, we demonstrate that one of these type IV secretion homologs, Cjp3/VirB10, is a glycoprotein. Treatment with various glycosidases and binding to soybean agglutinin indicated that the structure of the glycan present on VirB10 contains a terminal GalNAc, consistent with previous reports of N-linked glycans in
C. jejuni
. Site-directed mutagenesis of five putative N-linked glycosylation sites indicated that VirB10 is glycosylated at two sites, N32 and N97. Mutants in the N-linked general protein glycosylation (
pgl
) system of
C. jejuni
are significantly reduced in natural transformation, which is likely due, in part, to lack of glycosylation of VirB10. The natural transformation defect in a
virB10
mutant can be complemented in
trans
by using a plasmid expressing wild-type VirB10 or an N32A substitution but not by using a mutant expressing VirB10 with an N97A substitution. Taken together, these results suggest that glycosylation of VirB10 specifically at N97 is required for the function of the TFSS and for full competence in
C. jejuni
81-176.
Bifidobacterium longum
Requires a Fructokinase (Frk; ATP:
d
-Fructose 6-Phosphotransferase, EC 2.7.1.4) for Fructose Catabolism
Authors: Caescu Cristina I. , Vidal Olivier , Krzewinski Frédéric , Artenie Vlad , Bouquelet Stéphane 
Published: September 2004

ABSTRACT

Although the ability of
Bifidobacterium
spp. to grow on fructose as a unique carbon source has been demonstrated, the enzyme(s) needed to incorporate fructose into a catabolic pathway has hitherto not been defined. This work demonstrates that intracellular fructose is metabolized via the fructose-6-P phosphoketolase pathway and suggests that a fructokinase (Frk; EC 2.7.1.4) is the enzyme that is necessary and sufficient for the assimilation of fructose into this catabolic route in
Bifidobacterium longum
. The
B. longum
A10C fructokinase-encoding gene (
frk
) was expressed in
Escherichia coli
from a pET28 vector with an attached N-terminal histidine tag. The expressed enzyme was purified by affinity chromatography on a Co
2+
-based column, and the pH and temperature optima were determined. A biochemical analysis revealed that Frk displays the same affinity for fructose and ATP (

K
m

fructose
= 0.739 ± 0.18 mM and

K
m

ATP
= 0.756 ± 0.08 mM), is highly specific for
d
-fructose, and is inhibited by an excess of ATP (>12 mM). It was also found that
frk
is inducible by fructose and is subject to glucose-mediated repression. Consequently, this work presents the first characterization at the molecular and biochemical level of a fructokinase from a gram-positive bacterium that is highly specific for
d
-fructose.
Activation of the Rcs Signal Transduction System Is Responsible for the Thermosensitive Growth Defect of anEscherichia coliMutant Lacking Phosphatidylglycerol and Cardiolipin
Authors: Shiba Yasuhiro , Yokoyama Yasuko , Aono Yoshiko , Kiuchi Takashi , Kusaka Jin , Matsumoto Kouji , Hara Hiroshi 
Published: September 2004

ABSTRACTThe lethal effect of anEscherichia coli pgsAnull mutation, which causes a complete lack of the major acidic phospholipids, phosphatidylglycerol and cardiolipin, is alleviated by a lack of the major outer membrane lipoprotein encoded by thelppgene, but anlpp pgsAstrain shows a thermosensitive growth defect. Using transposon mutagenesis, we found that this thermosensitivity was suppressed by disruption of thercsC,rcsF, andyojNgenes, which code for a sensor kinase, accessory positive factor, and phosphotransmitter, respectively, of the Rcs phosphorelay signal transduction system initially identified as regulating the capsular polysaccharide synthesis (cps) genes. Disruption of thercsBgene coding for the response regulator of the system also suppressed the thermosensitivity, whereas disruption ofcpsEdid not. By monitoring the expression of acpsB′-lacfusion, we showed that the Rcs system is activated in thepgsAmutant and is reverted to a wild-type level by thercsmutations. These results indicate that envelope stress due to an acidic phospholipid deficiency activates the Rcs phosphorelay system and thereby causes the thermosensitive growth defect independent of the activation of capsule synthesis.
Towards Designer Cellulosomes in Clostridia: Mannanase Enrichment of the Cellulosomes Produced by
Clostridium cellulolyticum
Authors: Perret Stéphanie , Bélaich Anne , Fierobe Henri-Pierre , Bélaich Jean-Pierre , Tardif Chantal 
Published: September 2004

ABSTRACT

The
man5K
gene of
Clostridium cellulolyticum
was cloned and overexpressed in
Escherichia coli
. This gene encodes a 424-amino-acid preprotein composed of an N-terminal leader peptide, followed by a dockerin module and a C-terminal catalytic module belonging to family 5 of the glycosyl hydrolases. Mature Man5K displays 62% identity with ManA from
Clostridium cellulovorans
. Two forms of the protein were purified from
E. coli
; one form corresponds to the full-length enzyme (45 kDa), and a truncated form (39 kDa) lacks the N-terminal dockerin module. Both forms exhibit the same typical family 5 mannanase substrate preference; they are very active with the galactomannan locust bean gum, and the more galacto-substituted guar gum molecules are degraded less. The truncated form, however, displays fourfold-higher activity with galactomannans than the full-length enzyme. Man5K was successfully overproduced in
C. cellulolyticum
by using expression vectors. The
trans
-produced protein was found to be incorporated into the cellulosomes and became one of the major enzymatic components. Modified cellulosomes displayed 20-fold-higher specific activities than control fractions on galactomannan substrates, whereas the specific activity on crystalline cellulose was reduced by 20%. This work clearly showed that the composition of the cellulosomes is obviously regulated by the relative amounts of the enzymes produced and that this composition can be engineered in clostridia by structural gene cloning.
Two Chimeric Chromosomes ofStreptomyces coelicolorA3(2) Generated by Single Crossover of the Wild-Type Chromosome and Linear Plasmid SCP1
Authors: Yamasaki Masayuki , Kinashi Haruyasu 
Published: September 2004

ABSTRACTStreptomyces coelicolorA3(2) strain 2106 carries a 1.85-Mb linear plasmid, SCP1′-cysD, in addition to a 7.2-Mb linear chromosome. Macrorestriction analysis indicated that both linear DNAs are hybrids of the wild-type chromosome and the linear plasmid SCP1 on each side. Nucleotide sequencing of the fusion junctions revealed no homology between the recombination regions. SCP1′-cysDcontains an SCP1 telomere and a chromosomal telomere at each end and therefore does not have terminal inverted repeats. In addition, SCP1′-cysDcould not be eliminated from strain 2106 by various mutagenic treatments. Thus, we concluded that both the 7.2-Mb chromosome and SCP1′-cysDare chimeric chromosomes generated by a single crossover of the wild-type chromosome and SCP1. This may be regarded as a model of chromosomal duplication in genome evolution.
Complete Genomic Sequence of Bacteriophage B3, a Mu-Like Phage of
Pseudomonas aeruginosa
Authors: Braid Michael D. , Silhavy Jennifer L. , Kitts Christopher L. , Cano Raul J. , Howe Martha M. 
Published: September 2004

ABSTRACT

Bacteriophage B3 is a transposable phage of
Pseudomonas aeruginosa
. In this report, we present the complete DNA sequence and annotation of the B3 genome. DNA sequence analysis revealed that the B3 genome is 38,439 bp long with a G+C content of 63.3%. The genome contains 59 proposed open reading frames (ORFs) organized into at least three operons. Of these ORFs, the predicted proteins from 41 ORFs (68%) display significant similarity to other phage or bacterial proteins. Many of the predicted B3 proteins are homologous to those encoded by the early genes and head genes of Mu and Mu-like prophages found in sequenced bacterial genomes. Only two of the predicted B3 tail proteins are homologous to other well-characterized phage tail proteins; however, several Mu-like prophages and transposable phage D3112 encode approximately 10 highly similar proteins in their predicted tail gene regions. Comparison of the B3 genomic organization with that of Mu revealed evidence of multiple genetic rearrangements, the most notable being the inversion of the proposed B3 immunity/early gene region, the loss of Mu-like tail genes, and an extreme leftward shift of the B3 DNA modification gene cluster. These differences illustrate and support the widely held view that tailed phages are genetic mosaics arising by the exchange of functional modules within a diverse genetic pool.
Genome-Wide Molecular Clock and Horizontal Gene Transfer in Bacterial Evolution
Authors: Novichkov Pavel S. , Omelchenko Marina V. , Gelfand Mikhail S. , Mironov Andrei A. , Wolf Yuri I. , Koonin Eugene V. 
Published: September 2004

ABSTRACTWe describe a simple theoretical framework for identifying orthologous sets of genes that deviate from a clock-like model of evolution. The approach used is based on comparing the evolutionary distances within a set of orthologs to a standard intergenomic distance, which was defined as the median of the distribution of the distances between all one-to-one orthologs. Under the clock-like model, the points on a plot of intergenic distances versus intergenomic distances are expected to fit a straight line. A statistical technique to identify significant deviations from the clock-like behavior is described. For several hundred analyzed orthologous sets representing three well-defined bacterial lineages, the α-Proteobacteria, the γ-Proteobacteria, and theBacillus-Clostridiumgroup, the clock-like null hypothesis could not be rejected for ∼70% of the sets, whereas the rest showed substantial anomalies. Subsequent detailed phylogenetic analysis of the genes with the strongest deviations indicated that over one-half of these genes probably underwent a distinct form of horizontal gene transfer, xenologous gene displacement, in which a gene is displaced by an ortholog from a different lineage. The remaining deviations from the clock-like model could be explained by lineage-specific acceleration of evolution. The results indicate that although xenologous gene displacement is a major force in bacterial evolution, a significant majority of orthologous gene sets in three major bacterial lineages evolved in accordance with the clock-like model. The approach described here allows rapid detection of deviations from this mode of evolution on the genome scale.