bluuebunny/crossref_metadata_2025_split huggingface dataset:
Vaccination with Plasmid DNA Encoding TSA/LmSTI1 Leishmanial Fusion Proteins Confers Protection againstLeishmania majorInfection in Susceptible BALB/c Mice
Authors: Campos-Neto A. , Webb J. R. , Greeson K. , Coler R. N. , Skeiky Y. A. W. , Reed S. G. 
Published: July 2002

ABSTRACTWe have recently shown that a cocktail containing two leishmanial recombinant antigens (LmSTI1 and TSA) and interleukin-12 (IL-12) as an adjuvant induces solid protection in both a murine and a nonhuman primate model of cutaneous leishmaniasis. However, because IL-12 is difficult to prepare, is expensive, and does not have the stability required for a vaccine product, we have investigated the possibility of using DNA as an alternative means of inducing protective immunity. Here, we present evidence that the antigens TSA and LmSTI1 delivered in a plasmid DNA format either as single genes or in a tandem digene construct induce equally solid protection againstLeishmania majorinfection in susceptible BALB/c mice. Immunization of mice with either TSA DNA or LmSTI1 DNA induced specific CD4+-T-cell responses of the Th1 phenotype without a requirement for specific adjuvant. CD8 responses, as measured by cytotoxic-T-lymphocyte activity, were generated after immunization with TSA DNA but not LmSTI1 DNA. Interestingly, vaccination of mice with TSA DNA consistently induced protection to a much greater extent than LmSTI1 DNA, thus supporting the notion that CD8 responses might be an important accessory arm of the immune response for acquired resistance against leishmaniasis. Moreover, the protection induced by DNA immunization was specific for infection withLeishmania, i.e., the immunization had no effect on the course of infection of the mice challenged with an unrelated intracellular pathogen such asMycobacterium tuberculosis. Conversely, immunization of BALB/c mice with a plasmid DNA that is protective against challenge withM. tuberculosishad no effect on the course of infection of these mice withL. major. Together, these results indicate that the protection observed with the leishmanial DNA is mediated by acquired specific immune response rather than by the activation of nonspecific innate immune mechanisms. In addition, a plasmid DNA containing a fusion construct of the two genes was also tested. Similarly to the plasmids encoding individual proteins, the fusion construct induced both specific immune responses to the individual antigens and protection against challenge withL. major. These results confirm previous observations about the possibility of DNA immunization against leishmaniasis and lend support to the idea of using a single polygenic plasmid DNA construct to achieve polyspecific immune responses to several distinct parasite antigens.
Recruitment of Mammalian Cell Fibronectin to the Surface of
Chlamydia trachomatis
Authors: Kleba Betsy J. , Banta Erin , Lindquist Erika A. , Stephens Richard S. 
Published: July 2002

ABSTRACT

Pathogenic bacteria exploit the presence of various host cell molecules in order to colonize new tissues. Fibronectin is involved in a wide range of cell functions in vivo, and staphylococci, streptococci, and gonococci have evolved mechanisms to utilize this glycoprotein to mediate host cell binding. We show that elementary bodies (EB) from two biovars of
Chlamydia trachomatis
recruit fibronectin to their surfaces upon lysis of the host cell. We also demonstrate that a heparan sulfate lyase-sensitive molecule on chlamydial EB is responsible for binding at least a portion of this fibronectin.
An IS
257
-Derived Hybrid Promoter Directs Transcription of a
tetA
(K) Tetracycline Resistance Gene in the
Staphylococcus aureus
Chromosomal
mec
Region
Authors: Simpson Alice E. , Skurray Ronald A. , Firth Neville 
Published: July 2002

ABSTRACT

Transcription of the
tetA
(K) tetracycline resistance determinant encoded by an IS
257
-flanked cointegrated copy of a pT181-like plasmid, located within the chromosomal
mec
region of a methicillin-resistant
Staphylococcus aureus
isolate, has been investigated. The results demonstrated that transcription of
tetA
(K) in this strain is directed by both an IS
257
-derived hybrid promoter, which is stronger than the native
tetA
(K) promoter in the autonomous form of pT181, and a complete outwardly directed promoter identified within one end of IS
257
. Despite lower gene dosage, the chromosomal configuration was shown to afford a higher level of resistance than that mediated by pT181 in an autonomous multicopy state. Furthermore, competition studies revealed that a strain carrying the chromosomal
tetA
(K) determinant exhibited a higher level of fitness in the presence of tetracycline but not in its absence. This finding suggests that tetracycline has been a selective factor in the emergence of strains carrying a cointegrated pT181-like plasmid in their chromosomes. The results highlight the potential of IS
257
to influence the expression of neighboring genes, a property likely to enhance its capacity to mediate advantageous genetic rearrangements.
Characterization of the Ends and Target Sites of the Novel Conjugative Transposon Tn
5397
from
Clostridium difficile
: Excision and Circularization Is Mediated by the Large Resolvase, TndX
Authors: Wang Hongmei , Roberts Adam P. , Lyras Dena , Rood Julian I. , Wilks Mark , Mullany Peter 
Published: July 2002

ABSTRACT

Tn
5397
is a conjugative transposon that was originally isolated from
Clostridium difficile
. Previous analysis had shown that the central region of Tn
5397
was closely related to the conjugative transposon Tn
916
. However, in this work we obtained the DNA sequence of the ends of Tn
5397
and showed that they are completely different to those of Tn
916
. Tn
5397
did not contain the
int
and
xis
genes, which are required for the excision and integration of Tn
916
. Instead, the right end of Tn
5397
contained a gene,
tndX
, that appears to encode a member of the large resolvase family of site-specific recombinases. TndX is closely related to the TnpX resolvase from the mobilizable but nonconjugative chloramphenicol resistance transposons, Tn
4451
from
Clostridium perfringens
and Tn
4453
from
C. difficile
. Like the latter elements, inserted copies of Tn
5397
were flanked by a direct repeat of a GA dinucleotide. The Tn
5397
target sites were also shown to contain a central GA dinucleotide. Excision of the element in
C. difficile
completely regenerated the original target sequence. A circular form of the transposon, in which the left and right ends of the element were separated by a GA dinucleotide, was detected by PCR in both
Bacillus subtilis
and
C. difficile
. A Tn
5397
mutant in which part of
tndX
was deleted was constructed in
B. subtilis
. This mutant was nonconjugative and did not produce the circular form of Tn
5397
, indicating that the TndX resolvase has an essential role in the excision and transposition of Tn
5397
and is thus the first example of a member of the large resolvase family of recombinases being involved in conjugative transposon mobility. Finally, we showed that introduction of Tn
916
into a strain containing Tn
5397
induced the loss of the latter element in 95.6% of recipients.
Halocin S8: a 36-Amino-Acid Microhalocin from the Haloarchaeal Strain S8a
Authors: Price Lance B. , Shand Richard F. 
Published: July 2002

ABSTRACT

Halocin S8 is a hydrophobic microhalocin of 36 amino acids (3,580 Da) and is the first microhalocin to be described. This peptide antibiotic is unique since it is processed from inside a much larger, 33,962-Da pro-protein. Halocin S8 is quite robust, as it can be desalted, boiled, subjected to organic solvents, and stored at 4°C for extended periods without losing activity. The complete amino acid sequence of halocin S8 was obtained first by Edman degradation of the purified protein and verified from the
halS8
gene: H
2
N-S-D-C-N-I-N-S-N-T-A-A-D-V-I-L-C-F-N-Q-V-G-S-C-A-L-C-S-P-T-L-V-G-G-P-V-P-COOH. The
halS8
gene is encoded on an ∼200-kbp megaplasmid and contains a 933-bp open reading frame, of which 108 bp are occupied by halocin S8. Both the
halS8
promoter and the “leaderless”
halS8
transcript are typically haloarchaeal. Northern blot analysis revealed three
halS8
transcripts: two abundant and one minor. Inspection of the 3′ end of the gene showed only a single, weak termination site (5′-TTTAT-3′), suggesting that some processing of the larger transcripts may be involved. Expression of the
halS8
gene is growth stage dependent: basal
halS8
transcript levels are present in low concentrations during exponential growth but increase ninefold during the transition to stationary phase. Initially, halocin activity parallels
halS8
transcript levels very closely. However, when halocin activity plateaus, transcripts remain abundant, suggesting inhibition of translation at this point. Once the culture enters stationary phase, transcripts rapidly return to basal levels.
A DNA Ligase from a Hyperthermophilic Archaeon with Unique Cofactor Specificity
Authors: Nakatani Masaru , Ezaki Satoshi , Atomi Haruyuki , Imanaka Tadayuki 
Published: July 2002

ABSTRACT

A gene encoding DNA ligase (

lig
Tk

) from a hyperthermophilic archaeon,
Thermococcus kodakaraensis
KOD1, has been cloned and sequenced, and its protein product has been characterized.

lig
Tk

consists of 1,686 bp, corresponding to a polypeptide of 562 amino acids with a predicted molecular mass of 64,079 Da. Sequence comparison with previously reported DNA ligases and the presence of conserved motifs suggested that Lig

Tk

was an ATP-dependent DNA ligase. Phylogenetic analysis indicated that Lig

Tk

was closely related to the ATP-dependent DNA ligase from
Methanobacterium thermoautotrophicum
ΔH, a moderate thermophilic archaeon, along with putative DNA ligases from
Euryarchaeota
and
Crenarchaeota
. We expressed

lig
Tk

in
Escherichia coli
and purified the recombinant protein. Recombinant Lig

Tk

was monomeric, as is the case for other DNA ligases. The protein displayed DNA ligase activity in the presence of ATP and Mg
2+
. The optimum pH of Lig

Tk

was 8.0, the optimum concentration of Mg
2+
, which was indispensable for the enzyme activity, was 14 to 18 mM, and the optimum concentration of K
+
was 10 to 30 mM. Lig

Tk

did not display single-stranded DNA ligase activity. At enzyme concentrations of 200 nM, we observed significant DNA ligase activity even at 100°C. Unexpectedly, Lig

Tk

displayed a relatively small, but significant, DNA ligase activity when NAD
+
was added as the cofactor. Treatment of NAD
+
with hexokinase did not affect this activity, excluding the possibility of contaminant ATP in the NAD
+
solution. This unique cofactor specificity was also supported by the observation of adenylation of Lig

Tk

with NAD
+
. This is the first biochemical study of a DNA ligase from a hyperthermophilic archaeon.
Evolutionary Conservation of Methyl-Accepting Chemotaxis Protein Location in
Bacteria
and
Archaea
Authors: Gestwicki Jason E. , Lamanna Allison C. , Harshey Rasika M. , McCarter Linda L. , Kiessling Laura L. , Adler Julius 
Published: July 2002

ABSTRACT
The methyl-accepting chemotaxis proteins (MCPs) are concentrated at the cell poles in an evolutionarily diverse panel of bacteria and an archeon. In elongated cells, the MCPs are located both at the poles and at regions along the length of the cells. Together, these results suggest that MCP location is evolutionarily conserved.
Molecular Analysis of the
tagF
Gene, Encoding CDP-Glycerol:Poly(glycerophosphate) Glycerophosphotransferase of
Staphylococcus epidermidis
ATCC 14990
Authors: Fitzgerald Stephen N. , Foster Timothy J. 
Published: July 2002

ABSTRACT

Staphylococcus epidermidis
ATCC 14990 produces a wall-associated glycerol teichoic acid which is chemically identical to the major wall-associated teichoic acid of
Bacillus subtilis
168. The
S. epidermidis tagF
gene was cloned from genomic DNA and sequenced. When introduced on a plasmid vector into
B. subtilis
1A486 carrying the conditionally lethal temperature-sensitive mutation
tagF1
(
rodC1
), it expressed an 85-kDa protein which allowed colonies to grow at the restrictive temperature. This showed that the cloned
S. epidermidis
gene encodes a functional CDP-glycerol:poly(glycerophosphate) glycerophosphotransferase. An amino acid substitution at residue 616 in the recombinant TagF protein eliminated complementation. Unlike
B. subtilis
, where the
tagF
gene is part of the
tagDEF
operon, the
tagF
gene of
S. epidermidis
is not linked to any other
tag
genes. We attempted to disrupt the chromosomal
tagF
gene in
S. epidermidis
TU3298 by directed integration of a temperature-sensitive plasmid but this failed, whereas a control plasmid containing the 5′ end of
tagF
on a similarly sized DNA fragment was able to integrate. This suggests that the
tagF
gene is essential and that the TagF and other enzymes involved in teichoic acid biosynthesis could be targets for new antistaphylococcal drugs.
Utilization of Subsidiary Chromosomal Replication Terminators in
Bacillus subtilis
Authors: Griffiths A. A. , Wake R. G. 
Published: July 2002

ABSTRACT

The
Bacillus subtilis
merodiploid strain GSY1127 contains a large nontandem duplication of a portion of its chromosome within its left (anticlockwise) replication segment. This causes displacement of the replication terminus region to a noticeably asymmetric location relative to
oriC
. The utilization of the subsidiary replication terminators,
TerIII
and
TerV
, in the merodiploid strain has been compared with that in
B. subtilis
168. It is shown that
TerIII
is utilized to a significant extent in GSY1127 and that
TerV
is used only marginally at the most. Neither of these terminators is used to a measurable extent in the 168 strain. It is concluded that
TerIII
and
TerV
do indeed function as backups to the major terminator
TerI
, as has been generally thought. It is further concluded that, in the 168 strain, the vast majority of clockwise forks are arrested at the highly efficient
TerI
terminator, with fork fusion between the approaching forks occurring frequently while the clockwise fork is stationary at
TerI
.
A Single Amino Acid Substitution in a Mannosyltransferase, WbdA, Converts the
Escherichia coli
O9 Polysaccharide into O9a: Generation of a New O-Serotype Group
Authors: Kido Nobuo , Kobayashi Hidemitsu 
Published: July 2002

ABSTRACT

wbdA
is a mannosyltransferase gene that is involved in synthesis of the
Escherichia coli
O9a polysaccharide, a mannose homopolymer with a repeating unit of 2-αMan-1,2-αMan-1,3-αMan-1,3-αMan-1. The equivalent structural O polysaccharide in the
E. coli
O9 and
Klebsiella
O3 strains is 2-αMan-1,2-αMan-1,2-αMan-1,3-αMan-1,3-αMan-1, with an excess of one mannose in the 1,2 linkage. We have cloned
wbdA
genes from these O9 and O3 strains and shown by genetic and functional studies that
wbdA
is the only gene determining the O-polysaccharide structure of O9 or O9a. Based on functional analysis of chimeric genes and site-directed mutagenesis, we showed that a single amino acid substitution, C55R, in WbdA of
E. coli
O9 converts the O9 polysaccharide into O9a. DNA sequencing revealed the substitution to be conserved in other
E. coli
O9a strains. The reverse substitution, R55C, in WbdA of
E. coli
O9a resulted in lipopolysaccharide synthesis showing no ladder profile instead of the conversion of O9a to O9. This suggests that more than one amino acid substitution in WbdA is required for conversion from O9a to O9.
A Novel Sphingoglycolipid Containing Galacturonic Acid and 2-Hydroxy Fatty Acid in Cellular Lipids of
Sphingomonas yanoikuyae
Authors: Naka Takashi , Fujiwara Nagatoshi , Yabuuchi Eiko , Doe Matsumi , Kobayashi Kazuo , Kato Yoshiko , Yano Ikuya 
Published: July 2002

ABSTRACT

A novel sphingoglycolipid was isolated from
Sphingomonas yanoikuyae
, and its structure was identified as a galacturonosyl-β (1→1)-ceramide. This was a characteristic sphingoglycolipid present in
S. yanoikuyae
and certain other species of
Sphingomonas
, such as
Sphingomonas mali
,
Sphingomonas terrae
, and
Sphingomonas macrogoltabidus
, but not in the type species of
Sphingomonas
,
Sphingomonas paucimobilis
.
The N Terminus of FliM Is Essential To Promote Flagellar Rotation in
Rhodobacter sphaeroides
Authors: Poggio Sebastian , Osorio Aurora , Corkidi Gabriel , Dreyfus Georges , Camarena Laura 
Published: July 2002

ABSTRACT

FliM is part of the flagellar switch complex. Interaction of this protein with phospho-CheY (CheY-P) through its N terminus constitutes the main information relay point between the chemotactic system and the flagellum. In this work, we evaluated the role of the N terminus of FliM in the swimming behavior of
Rhodobacter sphaeroides
. Strains expressing the FliM protein with substitutions in residues previously reported in
Escherichia coli
as being important for interaction with CheY showed an increased stop frequency compared with wild-type cells. In accordance, we observed that
R. sphaeroides
cells expressing FliM lacking either the first 13 or 20 amino acids from the N terminus showed a stopped phenotype. We show evidence that FliMΔ13 and FliMΔ20 are stable proteins and that cells expressing them allow flagellin export at levels indistinguishable from those detected for the wild-type strain. These results suggest that the N-terminal region of FliM is required to promote swimming in this bacterium. The role of CheY in controlling flagellar rotation in this organism is discussed.
Autoamplification of a Two-Component Regulatory System Results in “Learning” Behavior
Authors: Hoffer Sally M. , Westerhoff Hans V. , Hellingwerf Klaas J. , Postma Pieter W. , Tommassen Jan 
Published: July 2002

ABSTRACT
We have tested the hypothesis that the autoamplification of two-component regulatory systems results in “learning” behavior, i.e., that bacteria respond faster or more extensively to a signal when a similar signal has been perceived in the past. Indeed, the induction of alkaline phosphatase activity upon phosphate limitation was faster if the cultures had been limited for phosphate previously, and this faster response correlated with the autoamplification of the cognate two-component system.
Roles of FrxA and RdxA Nitroreductases of
Helicobacter pylori
in Susceptibility and Resistance to Metronidazole
Authors: Jeong Jin-Yong , Mukhopadhyay Asish K. , Akada Junko K. , Dailidiene Daiva , Hoffman Paul S. , Berg Douglas E. 
Published: July 2002

ABSTRACT

The relative importance of the
frxA
and
rdxA
nitroreductase genes of
Helicobacter pylori
in metronidazole (MTZ) susceptibility and resistance has been controversial. Jeong et al. (J. Bacteriol. 182:5082–5090, 2000) had interpreted that Mtz
s
H. pylori
were of two types: type I, requiring only inactivation of
rdxA
to became resistant, and type II, requiring inactivation of both
rdxA
and
frxA
to become resistant;
frxA
inactivation by itself was not sufficient to confer resistance. In contrast, Kwon et al. (Antimicrob. Agents Chemother. 44:2133–2142, 2000) had interpreted that resistance resulted from inactivation either of
frxA
or
rdxA
. These two interpretations were tested here. Resistance was defined as efficient colony formation by single cells from diluted cultures rather than as growth responses of more dense inocula on MTZ-containing medium. Tests of three of Kwon's Mtz
s
strains showed that each was type II, requiring inactivation of both
rdxA
and
frxA
to become resistant. In additional tests, derivatives of
frxA
mutant strains recovered from MTZ-containing medium were found to contain new mutations in
rdxA
, and
frxA
inactivation slowed MTZ-induced killing of Mtz
s
strains. Northern blot analyses indicated that
frxA
mRNA, and perhaps also
rdxA
mRNA, were more abundant in type II than in type I strains. We conclude that development of MTZ resistance in
H. pylori
requires inactivation of
rdxA
alone or of both
rdxA
and
frxA
, depending on bacterial genotype, but rarely, if ever, inactivation of
frxA
alone, and that
H. pylori
strains differ in regulation of nitroreductase gene expression. We suggest that such regulatory differences may be significant functionally during human infection.
Hybrid Protein between Ribosomal Protein S16 and RimM of
Escherichia coli
Retains the Ribosome Maturation Function of Both Proteins
Authors: Lövgren J. Mattias , Wikström P. Mikael 
Published: July 2002

ABSTRACT

The RimM protein in
Escherichia coli
is associated with free 30S ribosomal subunits but not with 70S ribosomes and is important for efficient maturation of the 30S subunits. A mutant lacking RimM shows a sevenfold-reduced growth rate and a reduced translational efficiency. Here we show that a double alanine-for-tyrosine substitution in RimM prevents it from associating with the 30S subunits and reduces the growth rate of
E. coli
approximately threefold. Several faster-growing derivatives of the
rimM
amino acid substitution mutant were found that contain suppressor mutations which increased the amount of the RimM protein by two different mechanisms. Most of the suppressor mutations destabilized a secondary structure in the
rimM
mRNA, which previously was shown to decrease the synthesis of RimM by preventing the access of the ribosomes to the translation initiation region on the
rimM
mRNA. Three other independently isolated suppressor mutations created a fusion between
rpsP
, encoding the ribosomal protein S16, and
rimM
on the chromosome as a result of mutations in the
rpsP
stop codon preceding
rimM
. A severalfold-higher amount of the produced hybrid S16-RimM protein in the suppressor strains than of the native-sized RimM in the original substitution mutant seems to explain the suppression. The S16-RimM protein but not any native-size ribosomal protein S16 was found both in free 30S ribosomal subunits and in translationally active 70S ribosomes of the suppressor strains. This suggests that the hybrid protein can substitute for S16, which is an essential protein probably because of its role in ribosome assembly. Thus, the S16-RimM hybrid protein seems capable of carrying out the important functions that native S16 and RimM have in ribosome biogenesis.
Genetic and Biochemical Analyses of BvgA Interaction with the Secondary Binding Region of the
fha
Promoter of
Bordetella pertussis
Authors: Boucher Philip E. , Yang Mei-Shin , Schmidt Deanna M. , Stibitz Scott 
Published: July 2002

ABSTRACT

The BvgA-BvgS two-component signal transduction system regulates expression of virulence factors in
Bordetella pertussis
. The BvgA response regulator activates transcription by binding to target promoters, which include those for the genes encoding filamentous hemagglutinin (
fha
) and pertussis toxin (
ptx
). We have previously shown that at both promoters the phosphorylated form of BvgA binds multiple high- and low-affinity sites. Specifically, at the
fha
promoter, we proposed that there may be high- and a low-affinity binding sites for the BvgA dimer. In our present investigation, we used DNA binding analyses and in vitro and in vivo assays of promoters with substitutions and deletions to support and extend this hypothesis. Our observations indicate that (i) binding of BvgA∼P to a primary (high-affinity) site and a secondary binding region (lower affinity) is cooperative, (ii) although both the primary binding site and the secondary binding region are required for full activity of the wild-type (undeleted) promoter, deletion of two helical turns within the secondary binding region can produce a fully active or hyperactive promoter, and (iii) BvgA binding to the secondary binding region shows limited DNA sequence specificity.