bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparative Genetic Analysis of
Mycobacterium ulcerans
and
Mycobacterium marinum
Reveals Evidence of Recent Divergence
Authors: Stinear Timothy P. , Jenkin Grant A. , Johnson Paul D. R. , Davies John K. 
Published: July 2002

ABSTRACT

Previous studies of the 16S rRNA genes from
Mycobacterium ulcerans
and
Mycobacterium marinum
have suggested a very close genetic relationship between these species (99.6% identity). However, these organisms are phenotypically distinct and cause diseases with very different pathologies. To investigate this apparent paradox, we compared 3,306 nucleotides from the partial sequences of eight housekeeping and structural genes derived from 18
M. ulcerans
strains and 22
M. marinum
strains. This analysis confirmed the close genetic relationship inferred from the 16S rRNA data, with nucleotide sequence identity ranging from 98.1 to 99.7%. The multilocus sequence analysis also confirmed previous genotype studies of
M. ulcerans
that have identified distinct genotypes within a geographical region. Single isolates of both
M. ulcerans
and
M. marinum
that were shown by the sequence analysis to be the most closely related were then selected for further study. One- and two-dimensional pulsed-field gel electrophoresis was employed to compare the architecture and size of the genome from each species. Genome sizes of approximately 4.4 and 4.6 Mb were obtained for
M. ulcerans
and
M. marinum
, respectively. Significant macrorestriction fragment polymorphism was observed between the species. However, hybridization analysis of DNA cleaved with more frequently cutting enzymes identified significant preservation of the flanking sequence at seven of the eight loci sequenced. The exception was the 16S rRNA locus. Two high-copy-number insertion sequences, IS
2404
and IS
2606
, have recently been reported in
M. ulcerans
, and significantly, these elements are not present in
M. marinum
. Hybridization of the
Ase
I restriction fragments from
M. ulcerans
with IS
2404
and IS
2606
indicated widespread genome distribution for both of these repeated sequences. Taken together, these data strongly suggest that
M. ulcerans
has recently diverged from
M. marinum
by the acquisition and concomitant loss of DNA in a manner analogous to the emergence of
M. tuberculosis
, where species diversity is being driven mainly by the activity of mobile DNA elements.
Silencing and Activation of ClyA Cytotoxin Expression in
Escherichia coli
Authors: Westermark Marie , Oscarsson Jan , Mizunoe Yoshimitsu , Urbonaviciene Jurate , Uhlin Bernt Eric 
Published: July 2002

ABSTRACT

Cytolysin A (ClyA) is a pore-forming cytotoxic protein encoded by the
clyA
gene of
Escherichia coli
K-12. Genetic analysis suggested that
clyA
is silenced by the nucleoid protein H-NS. Purified H-NS protein showed preferential binding to
clyA
sequences in the promoter region, as evidenced by DNase I footprinting and gel mobility shift assays. Transcriptional derepression and activation of a chromosomal
clyA
::
luxAB
operon fusion were seen under conditions of H-NS deficiency and SlyA overproduction, respectively. In H-NS-deficient bacteria neither the absence nor the overproduction of SlyA affected the derepressed ClyA expression any further. Therefore, we suggest that overproduction of SlyA in

hns
+
E. coli

derepresses
clyA
transcription by counteracting H-NS. The cyclic AMP receptor protein (CRP) was required for ClyA expression, and it interacted with a predicted, albeit suboptimal, CRP binding site in the
clyA
upstream region. Site-specific alterations of the CRP binding site to match the consensus resulted in substantially higher levels of ClyA expression, while alterations that were predicted to reduce CRP binding reduced ClyA expression. During anaerobic growth the fumarate and nitrate reduction regulator (FNR) was important for ClyA expression, and the
clyA
gene could be activated by overexpression of FNR. A major
clyA
transcript having its 5′ end (+1) located 72 bp upstream of the translational start codon and 61 bp downstream of the CRP-FNR binding site was detected in the absence of H-NS. The
clyA
promoter was characterized as a class I promoter that could be transcriptionally activated by CRP and/or FNR. According to DNA bending analyses, the
clyA
promoter region has high intrinsic curvature. We suggest that it represents a regulatory region which is particularly susceptible to H-NS silencing, and its features are discussed in relation to regulation of other silenced operons.
Characterization of the Collagen-Binding S-Layer Protein CbsA of
Lactobacillus crispatus
Authors: Sillanpää Jouko , Martínez Beatriz , Antikainen Jenni , Toba Takahiro , Kalkkinen Nisse , Tankka Sanna , Lounatmaa Kari , Keränen Jaakko , Höök Magnus , Westerlund-Wikström Benita , Pouwels Peter H. , Korhonen Timo K. 
Published: July 2002

ABSTRACT

The
cbsA
gene of
Lactobacillus crispatus
strain JCM 5810, encoding a protein that mediates adhesiveness to collagens, was characterized and expressed in
Escherichia coli
. The
cbsA
open reading frame encoded a signal sequence of 30 amino acids and a mature polypeptide of 410 amino acids with typical features of a bacterial S-layer protein. The
cbsA
gene product was expressed as a His tag fusion protein, purified by affinity chromatography, and shown to bind solubilized as well as immobilized type I and IV collagens. Three other
Lactobacillus
S-layer proteins, SlpA, CbsB, and SlpnB, bound collagens only weakly, and sequence comparisons of CbsA with these S-layer proteins were used to select sites in
cbsA
where deletions and mutations were introduced. In addition, hybrid S-layer proteins that contained the N or the C terminus from CbsA, SlpA, or SlpnB as well as N- and C-terminally truncated peptides from CbsA were constructed by gene fusion. Analysis of these molecules revealed the major collagen-binding region within the N-terminal 287 residues and a weaker type I collagen-binding region in the C terminus of the CbsA molecule. The mutated or hybrid CbsA molecules and peptides that failed to polymerize into a periodic S-layer did not bind collagens, suggesting that the crystal structure with a regular array is optimal for expression of collagen binding by CbsA. Strain JCM 5810 was found to contain another S-layer gene termed
cbsB
that was 44% identical in sequence to
cbsA
. RNA analysis showed that
cbsA
, but not
cbsB
, was transcribed under laboratory conditions. S-layer-protein-expressing cells of strain JCM 5810 adhered to collagen-containing regions in the chicken colon, suggesting that CbsA-mediated collagen binding represents a true tissue adherence property of
L. crispatus
.
Characterization of SepL of Enterohemorrhagic
Escherichia coli
Authors: Kresse Andreas U. , Beltrametti Fabrizio , Müller Astrid , Ebel Frank , Guzmán Carlos A. 
Published: July 2002

ABSTRACT

The
sepL
gene is expressed in the locus of enterocyte effacement and therefore is most likely implicated in the attaching and effacing process, as are the products encoded by open reading frames located up- and downstream of this gene. In this study, the
sepL
gene of the enterohemorrhagic
Escherichia coli
(EHEC) strain EDL933 was analyzed and the corresponding polypeptide was characterized. We found that
sepL
is transcribed monocistronically and independently from the
esp
operon located downstream, which codes for the secreted proteins EspA, -D, and -B. Primer extension analysis allowed us to identify a single start of transcription 83 bp upstream of the
sepL
start codon. The analysis of the upstream regions led to the identification of canonical promoter sequences between positions −5 and −36. Translational fusions using
lacZ
as a reporter gene demonstrated that
sepL
is activated in the exponential growth phase by stimuli that are characteristic for the intestinal niche, e.g., a temperature of 37°C, a nutrient-rich environment, high osmolarity, and the presence of Mn
2+
. Protein localization studies showed that SepL was present in the cytoplasm and associated with the bacterial membrane fraction. To analyze the functional role of the SepL protein during infection of eukaryotic cells, an in-frame deletion mutant was generated. This
sepL
mutant was strongly impaired in its ability to attach to HeLa cells and induce a local accumulation of actin. These defects were partially restored by providing the
sepL
gene in
trans
. The EDL933Δ
sepL
mutant also exhibited an impaired secretion but not biosynthesis of Esp proteins, which was fully complemented by providing
sepL
in
trans
. These results demonstrate the crucial role played by SepL in the biological cycle of EHEC.
Terminal Oxidases of
Bacillus subtilis
Strain 168: One Quinol Oxidase, Cytochrome
aa
3
or Cytochrome
bd
, Is Required for Aerobic Growth
Authors: Winstedt Lena , von Wachenfeldt Claes 
Published: July 2002

ABSTRACT

The gram-positive endospore-forming bacterium
Bacillus subtilis
has, under aerobic conditions, a branched respiratory system comprising one quinol oxidase branch and one cytochrome oxidase branch. The system terminates in one of four alternative terminal oxidases. Cytochrome
caa
3
is a cytochrome
c
oxidase, whereas cytochrome
bd
and cytochrome
aa
3
are quinol oxidases. A fourth terminal oxidase, YthAB, is a putative quinol oxidase predicted from DNA sequence analysis. None of the terminal oxidases are, by themselves, essential for growth. However, one quinol oxidase (cytochrome
aa
3
or cytochrome
bd
) is required for aerobic growth of
B. subtilis
strain 168. Data indicating that cytochrome
aa
3
is the major oxidase used by exponentially growing cells in minimal and rich medium are presented. We show that one of the two heme-copper oxidases, cytochrome
caa
3
or cytochrome
aa
3
, is required for efficient sporulation of
B. subtilis
strain 168 and that deletion of YthAB in a strain lacking cytochrome
aa
3
makes the strain sporulation deficient.
Characterization of
Borrelia burgdorferi
BlyA and BlyB Proteins: a Prophage-Encoded Holin-Like System
Authors: Damman Christopher J. , Eggers Christian H. , Samuels D. Scott , Oliver Donald B. 
Published: July 2002

ABSTRACT

The conserved cp32 plasmid family of
Borrelia burgdorferi
was recently shown to be packaged into a bacteriophage particle (C. H. Eggers and D. S. Samuels, J. Bacteriol. 181:7308–7313, 1999). This plasmid encodes BlyA, a 7.4-kDa membrane-interactive protein, and BlyB, an accessory protein, which were previously proposed to comprise a hemolysis system. Our genetic and biochemical evidence suggests that this hypothesis is incorrect and that BlyA and BlyB function instead as a prophage-encoded holin or holin-like system for this newly described bacteriophage. An
Escherichia coli
mutant containing the
blyAB
locus that was defective for the normally cryptic host hemolysin SheA was found to be nonhemolytic, suggesting that induction of
sheA
by
blyAB
expression was responsible for the hemolytic activity observed previously. Analysis of the structural features of BlyA indicated greater structural similarity to bacteriophage-encoded holins than to hemolysins. Consistent with holin characteristics, subcellular localization studies with
E. coli
and
B. burgdorferi
indicated that BlyA is solely membrane associated and that BlyB is a soluble protein. Furthermore, BlyA exhibited a holin-like function by promoting the endolysin-dependent lysis of an induced lambda lysogen that was defective in the holin gene. Finally, induction of the cp32 prophage in
B. burgdorferi
dramatically stimulated
blyAB
expression. Our results provide the first evidence of a prophage-encoded holin within
Borrelia
.
Role of ς
B
in Adaptation of
Listeria monocytogenes
to Growth at Low Temperature
Authors: Becker Lynne A. , Evans Stefanie N. , Hutkins Robert W. , Benson Andrew K. 
Published: July 2002

ABSTRACT

The activity of ς
B
in
Listeria monocytogenes
is stimulated by high osmolarity and is necessary for efficient uptake of osmoprotectants. Here we demonstrate that, during cold shock, ς
B
contributes to adaptation in a growth phase-dependent manner and is necessary for efficient accumulation of betaine and carnitine as cryoprotectants.
Novel Phospholipase A Activity Secreted by
Legionella
Species
Authors: Flieger Antje , Gong Shimei , Faigle Marion , Deeg Martin , Bartmann Peter , Neumeister Birgid 
Published: July 2002

ABSTRACT

Bacterial phospholipases are regarded as a major virulence factor in infection. In bacteria associated with pneumonia, destruction of lung surfactant and host cell membranes by bacterial phospholipases secreted during infection is thought to contribute to the disease. Phospholipase C (PLC) activity has been described in several
Legionella
species (W. B. Baine, J. Gen. Microbiol. 134:489–498, 1988; W. B. Baine, J. Gen. Microbiol. 131:1383–1391, 1985). By using detection methods such as thin-layer chromatography and mass spectrometry, PLC activity could not be detected in several strains of
Legionella pneumophila
. Instead, phospholipid degradation was identified to be caused by a novel PLA activity. We could demonstrate that PLA secretion starts at the mid-exponential-growth phase when bacteria were grown in liquid culture. Several
Legionella
species secreted different amounts of PLA.
Legionella
PLA may act as a powerful agent in the mediation of pathogenicity due to destruction of lung surfactant and epithelial cells.
Capsule Biosynthesis and Basic Metabolism in
Streptococcus pneumoniae
Are Linked through the Cellular Phosphoglucomutase
Authors: Hardy Gail G. , Caimano Melissa J. , Yother Janet 
Published: July 2002

ABSTRACT

Synthesis of the type 3 capsular polysaccharide of
Streptococcus pneumoniae
requires UDP-glucose (UDP-Glc) and UDP-glucuronic acid (UDP-GlcUA) for production of the [3)-β-
d
-GlcUA-(1→4)-β-
d
-Glc-(1→]

n

polymer. The generation of UDP-Glc proceeds by conversion of Glc-6-P to Glc-1-P to UDP-Glc and is mediated by a phosphoglucomutase (PGM) and a Glc-1-P uridylyltransferase, respectively. Genes encoding both a Glc-1-P uridylyltransferase (
cps3U
) and a PGM homologue (
cps3M
) are present in the type 3 capsule locus, but these genes are not essential for capsule production. In this study, we characterized a mutant that produces fourfold less capsule than the type 3 parent. The spontaneous mutation resulting in this phenotype was not contained in the type 3 capsule locus but was instead located in a distant gene (
pgm
) encoding a second PGM homologue. The function of this gene product as a PGM was demonstrated through enzymatic and complementation studies. Insertional inactivation of
pgm
reduced capsule production to less than 10% of the parental level. The loss of PGM activity in the insertion mutants also caused growth defects and a strong selection for isolates containing second-site suppressor mutations. These results demonstrate that most of the PGM activity required for type 3 capsule biosynthesis is derived from the cellular PGM.
Characterization of Hydrogenase II from the Hyperthermophilic Archaeon
Pyrococcus furiosus
and Assessment of Its Role in Sulfur Reduction
Authors: Ma Kesen , Weiss Robert , Adams Michael W. W. 
Published: July 2002

ABSTRACT

The fermentative hyperthermophile
Pyrococcus furiosus
contains an NADPH-utilizing, heterotetrameric (αβγδ), cytoplasmic hydrogenase (hydrogenase I) that catalyzes both H
2
production and the reduction of elemental sulfur to H
2
S. Herein is described the purification of a second enzyme of this type, hydrogenase II, from the same organism. Hydrogenase II has an
M
r
of 320,000 ± 20,000 and contains four different subunits with
M
r
s of 52,000 (α), 39,000 (β), 30,000 (γ), and 24,000 (δ). The heterotetramer contained Ni (0.9 ± 0.1 atom/mol), Fe (21 ± 1.6 atoms/mol), and flavin adenine dinucleotide (FAD) (0.83 ± 0.1 mol/mol). NADPH and NADH were equally efficient as electron donors for H
2
production with

K
m

values near 70 μM and
k
cat
/

K
m

values near 350 min
−1
mM
−1
. In contrast to hydrogenase I, hydrogenase II catalyzed the H
2
-dependent reduction of NAD (

K
m

, 128 μM;
k
cat
/

K
m

, 770 min
−1
mM
−1
). Ferredoxin from
P. furiosus
was not an efficient electron carrier for either enzyme. Both H
2
and NADPH served as electron donors for the reduction of elemental sulfur (S
0
) and polysulfide by hydrogenase I and hydrogenase II, and both enzymes preferentially reduce polysulfide to sulfide rather than protons to H
2
using NADPH as the electron donor. At least two [4Fe-4S] and one [2Fe-2S] cluster were detected in hydrogenase II by electron paramagnetic resonance spectroscopy, but amino acid sequence analyses indicated a total of five [4Fe-4S] clusters (two in the β subunit and three in the δ subunit) and one [2Fe-2S] cluster (in the γ subunit), as well as two putative nucleotide-binding sites in the γ subunit which are thought to bind FAD and NAD(P)(H). The amino acid sequences of the four subunits of hydrogenase II showed between 55 and 63% similarity to those of hydrogenase I. The two enzymes are present in the cytoplasm at approximately the same concentration. Hydrogenase II may become physiologically relevant at low S
0
concentrations since it has a higher affinity than hydrogenase I for both S
0
and polysulfide.
Isolation and Characterization of
vicH
, Encoding a New Pleiotropic Regulator in
Vibrio cholerae
Authors: Tendeng Christian , Badaut Cyril , Krin Evelyne , Gounon Pierre , Ngo Saravuth , Danchin Antoine , Rimsky Sylvie , Bertin Philippe 
Published: July 2002

ABSTRACT

During the last decade, the
hns
gene and its product, the H-NS protein, have been extensively studied in
Escherichia coli
. H-NS-like proteins seem to be widespread in gram-negative bacteria. However, unlike in
E. coli
and in
Salmonella enterica
serovar Typhimurium, little is known about their role in the physiology of those organisms. In this report, we describe the isolation of
vicH
, an
hns
-like gene in
Vibrio cholerae
, the etiological agent of cholera. This gene was isolated from a
V. cholerae
genomic library by complementation of different phenotypes associated with an
hns
mutation in
E. coli
. It encodes a 135-amino-acid protein showing approximately 50% identity with both H-NS and StpA in
E. coli
. Despite a low amino acid conservation in the N-terminal part, VicH is able to cross-react with anti-H-NS antibodies and to form oligomers in vitro. The
vicH
gene is expressed as a single gene from two promoters in tandem and is induced by cold shock. A
V. cholerae
wild-type strain expressing a
vicHΔ92
gene lacking its 3′ end shows pleiotropic alterations with regard to mucoidy and salicin metabolism. Moreover, this strain is unable to swarm on semisolid medium. Similarly, overexpression of the
vicH
wild-type gene results in an alteration of swarming behavior. This suggests that VicH could be involved in the virulence process in
V. cholerae
, in particular by affecting flagellum biosynthesis.
OmpR Regulates the Stationary-Phase Acid Tolerance Response of
Salmonella enterica
Serovar Typhimurium
Authors: Bang Iel Soo , Kim Bae Hoon , Foster John W. , Park Yong Keun 
Published: July 2002

ABSTRACT

Tolerance to acidic environments is an important property of free-living and pathogenic enteric bacteria.
Salmonella enterica
serovar Typhimurium possesses two general forms of inducible acid tolerance. One is evident in exponentially growing cells exposed to a sudden acid shock. The other is induced when stationary-phase cells are subjected to a similar shock. These log-phase and stationary-phase acid tolerance responses (ATRs) are distinct in that genes identified as participating in log-phase ATR have little to no effect on the stationary-phase ATR (I. S. Lee, J. L. Slouczewski, and J. W. Foster, J. Bacteriol. 176:1422–1426, 1994). An insertion mutagenesis strategy designed to reveal genes associated with acid-inducible stationary-phase acid tolerance (stationary-phase ATR) yielded two insertions in the response regulator gene
ompR
. The
ompR
mutants were defective in stationary-phase ATR but not log-phase ATR. EnvZ, the known cognate sensor kinase, and the porin genes known to be controlled by OmpR,
ompC
and
ompF
, were not required for stationary-phase ATR. However, the alternate phosphodonor acetyl phosphate appears to play a crucial role in OmpR-mediated stationary-phase ATR and in the OmpR-dependent acid induction of
ompC
. This conclusion was based on finding that a mutant form of OmpR, which is active even though it cannot be phosphorylated, was able to suppress the acid-sensitive phenotype of an
ack pta
mutant lacking acetyl phosphate. The data also revealed that acid shock increases the level of
ompR
message and protein in stationary-phase cells. Thus, it appears that acid shock induces the production of OmpR, which in its phosphorylated state can trigger expression of genes needed for acid-induced stationary-phase acid tolerance.
Osmotic and Chill Activation of Glycine Betaine Porter II in
Listeria monocytogenes
Membrane Vesicles
Authors: Gerhardt Paul N. M. , Tombras Smith Linda , Smith Gary M. 
Published: July 2002

ABSTRACT

Listeria monocytogenes
is a foodborne pathogen known for its tolerance to conditions of osmotic and chill stress. Accumulation of glycine betaine has been found to be important in the organism's tolerance to both of these stresses. A procedure was developed for the purification of membranes from
L. monocytogenes
cells in which the putative ATP-driven glycine betaine permease glycine betaine porter II (Gbu) is functional. As is the case for the
L. monocytogenes
sodium-driven glycine betaine uptake system (glycine betaine porter I), uptake in this vesicle system was dependent on energization by ascorbate-phenazine methosulfate. Vesicles lacking the
gbu
gene product had no uptake activity. Transport by this porter did not require sodium ion and could be driven only weakly by artificial gradients. Uptake rates could be manipulated under conditions not affecting secondary transport but known to affect ATPase activity. The system was shown to be both osmotically activated and cryoactivated. Under conditions of osmotic activation, the system exhibited Arrhenius-type behavior although the uptake rates were profoundly affected by the physical state of the membrane, with breaks in Arrhenius curves at approximately 10 and 18°C. In the absence of osmotic activation, the permease could be activated by decreasing temperature within the range of 15 to 4°C. Kinetic analyses of the permease at 30°C revealed

K
m

values for glycine betaine of 1.2 and 2.9 μM with
V
max
values of 2,200 and 3,700 pmol/min · mg of protein under conditions of optimal osmotic activation as mediated by KCl and sucrose, respectively.