bluuebunny/crossref_metadata_2025_split huggingface dataset:
Organization, Structure, and Variability of the rRNA Operon of the Whipple's Disease Bacterium (
Tropheryma whippelii
)
Authors: Maiwald Matthias , von Herbay Axel , Lepp Paul W. , Relman David A. 
Published: July 2002

ABSTRACT

Whipple's disease is a systemic disorder associated with a cultivation-resistant, poorly characterized actinomycete,
Tropheryma whippelii
. We determined a nearly complete rRNA operon sequence of
T. whippelii
from specimens from 3 patients with Whipple's disease, as well as partial operon sequences from 43 patients. Variability was observed in the 16S-23S rRNA spacer sequences, leading to the description of five distinct sequence types. One specimen contained two spacer sequence types, raising the possibility of a double infection. Secondary structure models for the primary rRNA transcript and mature rRNAs revealed rare or unique features.
Sequential Inactivation of
rdxA
(HP0954) and
frxA
(HP0642) Nitroreductase Genes Causes Moderate and High-Level Metronidazole Resistance in
Helicobacter pylori
Authors: Jeong Jin-Yong , Mukhopadhyay Asish K. , Dailidiene Daiva , Wang Yipeng , Velapatiño Billie , Gilman Robert H. , Parkinson Alan J. , Nair G. Balakrish , Wong Benjamin C. Y. , Lam Shiu Kum , Mistry Rajesh , Segal Isidore , Yuan Yuan , Gao Hua , Alarcon Teresa , Brea MaNuel Lopez , Ito Yoshiyuki , Kersulyte Dangeruta , Lee Hae-Kyung , Gong Yan , Goodwin Avery , Hoffman Paul S. , Berg Douglas E. 
Published: July 2002

ABSTRACT

Helicobacter pylori
is a human-pathogenic bacterial species that is subdivided geographically, with different genotypes predominating in different parts of the world. Here we test and extend an earlier conclusion that metronidazole (Mtz) resistance is due to mutation in
rdxA
(HP0954), which encodes a nitroreductase that converts Mtz from prodrug to bactericidal agent. We found that (i)
rdxA
genes PCR amplified from 50 representative Mtz
r
strains from previously unstudied populations in Asia, South Africa, Europe, and the Americas could, in each case, transform Mtz
s
H. pylori
to Mtz
r
; (ii) Mtz
r
mutant derivatives of a cultured Mtz
s
strain resulted from mutation in
rdxA
; and (iii) transformation of Mtz
s
strains with
rdxA
-null alleles usually resulted in moderate level Mtz resistance (16 μg/ml). However, resistance to higher Mtz levels was common among clinical isolates, a result that implicates at least one additional gene. Expression in
Escherichia coli
of
frxA
(HP0642; flavin oxidoreductase), an
rdxA
paralog, made this normally resistant species Mtz
s
, and
frxA
inactivation enhanced Mtz resistance in
rdxA
-deficient cells but had little effect on the Mtz susceptibility of
rdxA
+
cells. Strains carrying
frxA
-null and
rdxA
-null alleles could mutate to even higher resistance, a result implicating one or more additional genes in residual Mtz susceptibility and hyperresistance. We conclude that most Mtz resistance in
H. pylori
depends on
rdxA
inactivation, that mutations in
frxA
can enhance resistance, and that genes that confer Mtz resistance without
rdxA
inactivation are rare or nonexistent in
H. pylori
populations.
Functional Organization and Insertion Specificity of IS607, a Chimeric Element ofHelicobacter pylori
Authors: Kersulyte Dangeruta , Mukhopadhyay Asish K. , Shirai Mutsinori , Nakazawa Teruko , Berg Douglas E. 
Published: July 2002

ABSTRACTA search by subtractive hybridization for sequences present in only certain strains ofHelicobacter pyloriled to the discovery of a 2-kb transposable element to be called IS607, which further PCR and hybridization tests indicated was present in about one-fifth ofH. pyloristrains worldwide. IS607contained two open reading frames (ORFs) of possibly different phylogenetic origin. One ORF (orfB) exhibited protein-level homology to one of two putative transposase genes found in several other chimeric elements including IS605(also ofH. pylori) and IS1535(ofMycobacterium tuberculosis). The second IS607gene (orfA) was unrelated to the second gene of IS605and might possibly be chimeric itself: it exhibited protein-level homology tomerRbacterial regulatory genes in the first ∼50 codons and homology to the second gene of IS1535(annotated as “resolvase,” apparently due to a weak short recombinase motif) in the remaining three-fourths of its length. IS607was found to transpose inEscherichia coli, and analyses of sequences of IS607-target DNA junctions inH. pyloriandE. coliindicated that it inserted either next to or between adjacent GG nucleotides, and generated either a 2-bp or a 0-bp target sequence duplication, respectively. Mutational tests showed that its transposition inE. colirequiredorfAbut notorfB, suggesting that OrfA protein may represent a new, previously unrecognized, family of bacterial transposases.
Novel Role for an HPt Domain in Stabilizing the Phosphorylated State of a Response Regulator Domain
Authors: Janiak-Spens Fabiola , Sparling David P. , West Ann H. 
Published: July 2002

ABSTRACT

Two-component regulatory systems that utilize a multistep phosphorelay mechanism often involve a histidine-containing phosphotransfer (HPt) domain. These HPt domains serve an essential role as histidine-phosphorylated protein intermediates during phosphoryl transfer from one response regulator domain to another. In
Saccharomyces cerevisiae
, the YPD1 protein facilitates phosphoryl transfer from a hybrid sensor kinase, SLN1, to two distinct response regulator proteins, SSK1 and SKN7. Because the phosphorylation state largely determines the functional state of response regulator proteins, we have carried out a comparative study of the phosphorylated lifetimes of the three response regulator domains associated with SLN1, SSK1, and SKN7 (R1, R2, and R3, respectively). The isolated regulatory domains exhibited phosphorylated lifetimes within the range previously observed for other response regulator domains (i.e., several minutes to several hours). However, in the presence of YPD1, we found that the half-life of phosphorylated SSK1-R2 was dramatically extended (almost 200-fold longer than in the absence of YPD1). This stabilization effect was specific for SSK1-R2 and was not observed for SLN1-R1 or SKN7-R3. Our findings suggest a mechanism by which SSK1 is maintained in its phosphorylated state under normal physiological conditions and demonstrate an unprecedented regulatory role for an HPt domain in a phosphorelay signaling system.
Cloning and Functional Characterization of an NAD
+
-Dependent DNA Ligase from
Staphylococcus aureus
Authors: Kaczmarek Frank S. , Zaniewski Richard P. , Gootz Thomas D. , Danley Dennis E. , Mansour Mahmoud N. , Griffor Matt , Kamath Ajith V. , Cronan Melissa , Mueller John , Sun Dongxu , Martin Patrick K. , Benton Bret , McDowell Laura , Biek Donald , Schmid Molly B. 
Published: July 2002

ABSTRACT

A
Staphylococcus aureus
mutant conditionally defective in DNA ligase was identified by isolation of complementing plasmid clones that encode the
S. aureus lig
A gene. Orthologues of the putative
S. aureus
NAD
+
-dependent DNA ligase could be identified in the genomes of
Bacillus stearothermophilus
and other gram-positive bacteria and confirmed the presence of four conserved amino acid motifs, including motif I, KXDG with lysine 112, which is believed to be the proposed site of adenylation. DNA sequence comparison of the
ligA
genes from wild type and temperature-sensitive
S. aureus
strain NT64 identified a single base alteration that is predicted to result in the amino acid substitution E46G. The
S. aureus ligA
gene was cloned and overexpressed in
Escherichia coli
, and the enzyme was purified to near homogeneity. NAD
+
-dependent DNA ligase activity was demonstrated with the purified enzyme by measuring ligation of
32
P-labeled 30-mer and 29-mer oligonucleotides annealed to a complementary strand of DNA. Limited proteolysis of purified
S. aureus
DNA ligase by thermolysin produced products with apparent molecular masses of 40, 22, and 21 kDa. The fragments were purified and characterized by N-terminal sequencing and mass analysis. The N-terminal fragment (40 kDa) was found to be fully adenylated. A fragment from residues 1 to 315 was expressed as a His-tagged fusion in
E. coli
and purified for functional analysis. Following deadenylation with nicotinamide mononucleotide, the purified fragment could self-adenylate but lacked detectable DNA binding activity. The 21- and 22-kDa C-terminal fragments, which lacked the last 76 amino acids of the DNA ligase, had no adenylation activity or DNA binding activity. The intact 30-kDa C terminus of the
S. aureus
LigA protein expressed in
E. coli
did demonstrate DNA binding activity. These observations suggest that, as in the case with the NAD
+
-dependent DNA ligase from
B. stearothermophilus
, two independent functional domains exist in
S. aureus
DNA ligase, consisting of separate adenylation and DNA binding activities. They also demonstrate a role for the extreme C terminus of the ligase in DNA binding. As there is much evidence to suggest that DNA ligase is essential for bacterial survival, its discovery in the important human pathogen
S. aureus
indicates its potential as a broad-spectrum antibacterial target for the identification of novel antibiotics.
Purification, Characterization, and Genetic Analysis of Cu-Containing Dissimilatory Nitrite Reductase from a Denitrifying Halophilic Archaeon,
Haloarcula marismortui
Authors: Ichiki Hirotaka , Tanaka Yoko , Mochizuki Kiyotaka , Yoshimatsu Katsuhiko , Sakurai Takeshi , Fujiwara Taketomo 
Published: July 2002

ABSTRACT

Cu-containing dissimilatory nitrite reductase (CuNiR) was purified from denitrifying cells of a halophilic archaeon,
Haloarcula marismortui
. The purified CuNiR appeared blue in the oxidized state, possessing absorption peaks at 600 and 465 nm in the visible region. Electron paramagnetic resonance spectroscopy suggested the presence of type 1 Cu (g
II
= 2.232; A
II
= 4.4 mT) and type 2 Cu centers (g
II
= 2.304; A
II
= 13.3 mT) in the enzyme. The enzyme contained two subunits, whose apparent molecular masses were 46 and 42 kDa, according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal amino acid sequence analysis indicated that the two subunits were identical, except that the 46-kDa subunit was 16 amino acid residues longer than the 42-kDa subunit in the N-terminal region. A
nirK
gene encoding the CuNiR was cloned and sequenced, and the deduced amino acid sequence with a residual length of 361 amino acids was homologous (30 to 41%) with bacterial counterparts. Cu-liganding residues His-133, Cys-174, His-182, and Met-187 (for type 1 Cu) and His-138, His-173, and His-332 (for type 2 Cu) were conserved in the enzyme. As generally observed in the halobacterial enzymes, the enzymatic activity of the purified CuNiR was enhanced during increasing salt concentration and reached its maximum in the presence of 2 M NaCl with the value of 960 μM NO
2
−
· min
−1
· mg
−1
.
uvrA
Is an Acid-Inducible Gene Involved in the Adaptive Response to Low pH in
Streptococcus mutans
Authors: Hanna Michael N. , Ferguson Ronald J. , Li Yung-Hua , Cvitkovitch Dennis G. 
Published: July 2002

ABSTRACT

The pH-inducible acid tolerance response (ATR) is believed to play a major role in acid adaptation and virulence of
Streptococcus mutans
. To study this phenomenon in
S
.
mutans
JH1005, differential display PCR was used to identify and clone 13 cDNA products that had increased expression in response to pH 5.0 compared to that of pH 7.5-grown cells. One of these products, confirmed to be pH inducible by RNA dot blot and reverse transcription-PCR analyses, had 67% identity to a
uvrA
-UV repair excinuclease gene in
Bacillus subtilis
. Further sequence analysis of the
uvrA
homologue using the
S
.
mutans
genome database revealed that the complete gene was encoded in an open reading frame (ORF) of 2,829 bp (944 amino acids; 104.67 kDa). Immediately 3′ of
uvrA
was an ORF encoding a putative aminopeptidase gene (
pepP
).
uvrA
knockouts were constructed in
S
.
mutans
strains JH1005, NG8, and UA159 using allelic-exchange mutagenesis, replacing the entire gene with an erythromycin resistance cassette. As with
uvrA
mutants in other bacteria, the
S
.
mutans uvrA
mutants were extremely sensitive to UV irradiation. The
uvrA
mutant of
S
.
mutans
JH1005 was also more sensitive than the wild type to growth at pH 5.0, showing a 15% reduction in growth rate and a 14% reduction in final resting culture density. Acid-adapted
S
.
mutans
JH1005
uvrA
mutants were shown to be more resistant to UV irradiation than was the parent but were unable to survive exposure to a killing pH of 3.0. Moreover, agarose gel electrophoretic analysis of chromosomal DNA isolated from
uvrA
-deficient cells exposed to low pH demonstrated more DNA damage than that for the wild-type strain. Here we suggest that
uvrA
and the nucleotide excision repair pathway are involved in the repair of acid-induced DNA damage and are associated with successful adaptation of
S
.
mutans
to low pH.
T7 Single Strand DNA Binding Protein but Not T7 Helicase Is Required for DNA Double Strand Break Repair
Authors: Yu Man , Masker Warren 
Published: July 2002

ABSTRACT

An in vitro system based on
Escherichia coli
infected with bacteriophage T7 was used to test for involvement of host and phage recombination proteins in the repair of double strand breaks in the T7 genome. Double strand breaks were placed in a unique
Xho
I site located approximately 17% from the left end of the T7 genome. In one assay, repair of these breaks was followed by packaging DNA recovered from repair reactions and determining the yield of infective phage. In a second assay, the product of the reactions was visualized after electrophoresis to estimate the extent to which the double strand breaks had been closed. Earlier work demonstrated that in this system double strand break repair takes place via incorporation of a patch of DNA into a gap formed at the break site. In the present study, it was found that extracts prepared from uninfected
E. coli
were unable to repair broken T7 genomes in this in vitro system, thus implying that phage rather than host enzymes are the primary participants in the predominant repair mechanism. Extracts prepared from an
E. coli recA
mutant were as capable of double strand break repair as extracts from a wild-type host, arguing that the
E. coli
recombinase is not essential to the recombinational events required for double strand break repair. In T7 strand exchange during recombination is mediated by the combined action of the helicase encoded by gene 4 and the annealing function of the gene 2.5 single strand binding protein. Although a deficiency in the gene 2.5 protein blocked double strand break repair, a gene 4 deficiency had no effect. This argues that a strand transfer step is not required during recombinational repair of double strand breaks in T7 but that the ability of the gene 2.5 protein to facilitate annealing of complementary single strands of DNA is critical to repair of double strand breaks in T7.
Link between the Membrane-Bound Pyridine Nucleotide Transhydrogenase and Glutathione-Dependent Processes in
Rhodobacter sphaeroides
Authors: Hickman Jason W. , Barber Robert D. , Skaar Eric P. , Donohue Timothy J. 
Published: July 2002

ABSTRACT

The presence of a glutathione-dependent pathway for formaldehyde oxidation in the facultative phototroph
Rhodobacter sphaeroides
has allowed the identification of gene products that contribute to formaldehyde metabolism. Mutants lacking the glutathione-dependent formaldehyde dehydrogenase (GSH-FDH) are sensitive to metabolic sources of formaldehyde, like methanol. This growth phenotype is correlated with a defect in formaldehyde oxidation. Additional methanol-sensitive mutants were isolated that contained Tn
5
insertions in
pntA
, which encodes the α subunit of the membrane-bound pyridine nucleotide transhydrogenase. Mutants lacking transhydrogenase activity have phenotypic and physiological characteristics that are different from those that lack GSH-FDH activity. For example, cells lacking transhydrogenase activity can utilize methanol as a sole carbon source in the absence of oxygen and do not display a formaldehyde oxidation defect, as determined by whole-cell
13
C-nuclear magnetic resonance. Since transhydrogenase can be a major source of NADPH, loss of this enzyme could result in a requirement for another source for this compound. Evidence supporting this hypothesis includes increased specific activities of other NADPH-producing enzymes and the finding that glucose utilization by the Entner-Doudoroff pathway restores aerobic methanol resistance to cells lacking transhydrogenase activity. Mutants lacking transhydrogenase activity also have higher levels of glutathione disulfide under aerobic conditions, so it is consistent that this strain has increased sensitivity to oxidative stress agents like diamide, which are known to alter the oxidation reduction state of the glutathione pool. A model will be presented to explain the role of transhydrogenase under aerobic conditions when cells need glutathione both for GSH-FDH activity and to repair oxidatively damaged proteins.
Phylogeny and Functional Expression of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from the Autotrophic Ammonia-Oxidizing Bacterium
Nitrosospira
sp.Isolate 40KI
Authors: Utåker Janne B. , Andersen Kjell , Aakra Ågot , Moen Birgitte , Nes Ingolf F. 
Published: July 2002

ABSTRACT

The autotrophic ammonia-oxidizing bacteria (AOB), which play an important role in the global nitrogen cycle, assimilate CO
2
by using ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). Here we describe the first detailed study of RubisCO (
cbb
) genes and proteins from the AOB. The
cbbLS
genes from
Nitrosospira
sp. isolate 40KI were cloned and sequenced. Partial sequences of the RubisCO large subunit (CbbL) from 13 other AOB belonging to the β and γ subgroups of the class
Proteobacteria
are also presented. All except one of the β-subgroup AOB possessed a red-like type I RubisCO with high sequence similarity to the
Ralstonia eutropha
enzyme. All of these new red-like RubisCOs had a unique six-amino-acid insert in CbbL. Two of the AOB,
Nitrosococcus halophilus
Nc4 and
Nitrosomonas europaea
Nm50, had a green-like RubisCO. With one exception, the phylogeny of the AOB CbbL was very similar to that of the 16S rRNA gene. The presence of a green-like RubisCO in
N. europaea
was surprising, as all of the other β-subgroup AOB had red-like RubisCOs. The green-like enzyme of
N. europaea
Nm50 was probably acquired by horizontal gene transfer. Functional expression of
Nitrosospira
sp. isolate 40KI RubisCO in the chemoautotrophic host
R. eutropha
was demonstrated. Use of an expression vector harboring the
R. eutropha cbb
control region allowed regulated expression of
Nitrosospira
sp. isolate 40KI RubisCO in an
R. eutropha cbb
deletion strain. The
Nitrosospira
RubisCO supported autotrophic growth of
R. eutropha
with a doubling time of 4.6 h. This expression system may allow further functional analysis of AOB
cbb
genes.
Speed versus Efficiency in Microbial Growth and the Role of Parallel Pathways
Authors: Helling Robert B. 
Published: July 2002

ABSTRACTMany microorganisms have sets of parallel pathways for ATP production in respiration and for ATP utilization in glutamate synthesis. The alternatives differ in efficiency of ATP production and utilization. The choice among these parallel pathways has been hypothesized to control the speed and efficiency of growth. Thus, the organism should be able to alleviate (or exaggerate) deficiency in one pathway by deleting another. I show here that inEscherichia colithe effect of lack of the glutamate-synthesizing enzyme glutamate dehydrogenase on glucose-limited growth is altered predictably byndh,cyo, andcydmutations affecting parallel pathways leading to ATP synthesis in respiration.
Mobility of a Restriction-Modification System Revealed by Its Genetic Contexts in Three Hosts
Authors: Naderer Marc , Brust Jessica R. , Knowle Dieter , Blumenthal Robert M. 
Published: July 2002

ABSTRACT

The flow of genes among prokaryotes plays a fundamental role in shaping bacterial evolution, and restriction-modification systems can modulate this flow. However, relatively little is known about the distribution and movement of restriction-modification systems themselves. We have isolated and characterized the genes for restriction-modification systems from two species of
Salmonella
,
S. enterica serovar
Paratyphi A and
S. enterica
serovar Bareilly. Both systems are closely related to the
Pvu
II restriction-modification system and share its target specificity. In the case of
S. enterica
serovar Paratyphi A, the restriction endonuclease is inactive, apparently due to a mutation in the subunit interface region. Unlike the chromosomally located
Salmonella
systems, the
Pvu
II system is plasmid borne. We have completed the sequence characterization of the
Pvu
II plasmid pPvu1, originally from
Proteus vulgaris
, making this the first completely sequenced plasmid from the genus
Proteus
. Despite the pronounced similarity of the three restriction-modification systems, the flanking sequences in
Proteus
and
Salmonella
are completely different. The
Spt
AI and
Sba
I genes lie between an equivalent pair of bacteriophage P4-related open reading frames, one of which is a putative integrase gene, while the
Pvu
II genes are adjacent to a
mob
operon and a XerCD recombination (
cer
) site.
Variation in the Spacer Regions Separating tRNA Genes in
Renibacterium salmoninarum
Distinguishes Recent Clinical Isolates from the Same Location
Authors: Alexander Sarah M. , Grayson T. Hilton , Chambers Edel M. , Cooper Lynne F. , Barker Gavin A. , Gilpin Martyn L. 
Published: July 2002

ABSTRACT

A means for distinguishing between clinical isolates of
Renibacterium salmoninarum
that is based on the PCR amplification of length polymorphisms in the tRNA intergenic spacer regions (tDNA-ILPs) was investigated. The method used primers specific to nucleotide sequences of
R. salmoninarum
tRNA genes and tRNA intergenic spacer regions that had been generated by using consensus tRNA gene primers. Twenty-one PCR products were sequenced from five isolates of
R. salmoninarum
from the United States, England, and Scotland, and four complete tRNA genes and spacer regions were identified. Sixteen specific PCR primers were designed and tested singly and in all possible pairwise combinations for their potential to discriminate between isolates from recent clinical outbreaks of bacterial kidney disease (BKD) in the United Kingdom. Fourteen of the isolates were cultured from kidney samples taken from fish displaying clinical signs of BKD on five farms, and some of the isolates came from the same farm and at the same time. The tDNA-ILP profiles separated 22 clinical isolates into nine groups and highlighted that some farms may have had more than one source of infection. The grouping of isolates improved on the discriminatory power of previously reported typing methods based on randomly amplified polymorphic DNA analysis and restriction fragment length profiles developed using insertion sequence IS
994
. Our method enabled us to make divisions between closely related clinical isolates of
R. salmoninarum
that have identical exact tandem repeat (ETR-A) loci, rRNA intergenic spacer sequences, and IS
994
profiles.