bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Hydrogen Peroxide-Forming NADH Oxidase That Functions as an Alkyl Hydroperoxide Reductase in
Amphibacillus xylanus
Authors: Niimura Youichi , Nishiyama Yoshitaka , Saito Daisuke , Tsuji Hirokazu , Hidaka Makoto , Miyaji Tatsurou , Watanabe Toshiro , Massey Vincent 
Published: July 2002

ABSTRACT

The
Amphibacillus xylanus
NADH oxidase, which catalyzes the reduction of oxygen to hydrogen peroxide with β-NADH, can also reduce hydrogen peroxide to water in the presence of free flavin adenine dinucleotide (FAD) or the small disulfide-containing
Salmonella enterica
AhpC protein. The enzyme has two disulfide bonds, Cys128-Cys131 and Cys337-Cys340, which can act as redox centers in addition to the enzyme-bound FAD (K. Ohnishi, Y. Niimura, M. Hidaka, H. Masaki, H. Suzuki, T. Uozumi, and T. Nishino, J. Biol. Chem. 270:5812–5817, 1995). The NADH-FAD reductase activity was directly dependent on the FAD concentration, with a second-order rate constant of approximately 2.0 × 10
6
M
−1
s
−1
. Rapid-reaction studies showed that the reduction of free flavin occurred through enzyme-bound FAD, which was reduced by NADH. The peroxidase activity of NADH oxidase in the presence of FAD resulted from reduction of peroxide by free FADH
2
reduced via enzyme-bound FAD. This peroxidase activity was markedly decreased in the presence of oxygen, since the free FADH
2
is easily oxidized by oxygen, indicating that this enzyme system is unlikely to be functional in aerobic growing cells. The
A. xylanus ahpC
gene was cloned and overexpressed in
Escherichia coli
. When the NADH oxidase was coupled with
A. xylanus
AhpC, the peroxidase activity was not inhibited by oxygen. The
V
max
values for hydrogen peroxide and cumene hydroperoxide reduction were both approximately 150 s
−1
. The

K
m

values for hydrogen peroxide and cumene hydroperoxide were too low to allow accurate determination of their values. Both AhpC and NADH oxidase were induced under aerobic conditions, a clear indication that these proteins are involved in the removal of peroxides under aerobic growing conditions.
Proteolysis of the McpA Chemoreceptor Does Not Require the
Caulobacter
Major Chemotaxis Operon
Authors: Tsai Jeng-Wen , Alley M. R. K. 
Published: July 2002

ABSTRACT

The degradation of the McpA chemoreceptor in
Caulobacter crescentus
accompanies the swarmer cell to the stalked-cell differentiation event. To further analyze the requirements for its degradation, we have constructed a series of strains that have deletions in the
mcpA
gene and in the
mcpA
chemotaxis operon. Internal deletions of the
mcpA
gene demonstrate that the highly conserved domain (signalling unit) and the methylation domains are not required for cell cycle-regulated proteolysis. The deletion of the chemotaxis operon, which is absolutely required for chemotaxis and McpA chemoreceptor methylation, has no effect on McpA proteolysis.
Comparison of the UDP-
N
-Acetylmuramate:
l
-Alanine Ligase Enzymes from
Mycobacterium tuberculosis
and
Mycobacterium leprae
Authors: Mahapatra Sebabrata , Crick Dean C. , Brennan Patrick J. 
Published: July 2002

ABSTRACT

In the peptidoglycan of
Mycobacterium leprae
,
l
-alanine of the side chain is replaced by glycine. When expressed in
Escherichia coli
, MurC (UDP-
N
-acetyl-muramate:
l
-alanine ligase) of
M. leprae
showed

K
m

and
V
max
for
l
-alanine and glycine similar to those of
Mycobacterium tuberculosis
MurC, suggesting that another explanation should be sought for the presence of glycine.
Catabolite Repression Control by Crc in 2xYT Medium Is Mediated by Posttranscriptional Regulation of
bkdR
Expression in
Pseudomonas putida
Authors: Hester Kathryn L. , Madhusudhan K. T. , Sokatch John R. 
Published: July 2002

ABSTRACT

The effect of growth in 2xYT medium on catabolite repression control in
Pseudomonas putida
has been investigated using the
bkd
operon, encoding branched-chain keto acid dehydrogenase. Crc (catabolite repression control protein) was shown to be responsible for repression of
bkd
operon transcription in 2xYT. BkdR levels were elevated in a
P. putida crc
mutant, but
bkdR
transcript levels were the same in both wild type and
crc
mutant. This suggests that the mechanism of catabolite repression control in rich media by Crc involves posttranscriptional regulation of the bkdR message.
Regulation of Cold Shock-Induced RNA Helicase Gene Expression in the Cyanobacterium
Anabaena
sp. Strain PCC 7120
Authors: Chamot Danuta , Owttrim George W. 
Published: July 2002

ABSTRACT

Expression of the
Anabaena
sp. strain PCC 7120 RNA helicase gene
crhC
is induced by cold shock.
crhC
transcripts are not detectable at 30°C but accumulate at 20°C, and levels remain elevated for the duration of the cold stress. Light-derived metabolic capability, and not light per se, is required for
crhC
transcript accumulation. Enhanced
crhC
mRNA stability contributes significantly to the accumulation of
crhC
transcripts, with the
crhC
half-life increasing sixfold at 20°C. The accumulation is reversible, with the cells responding more rapidly to temperature downshifts than to upshifts, as a result of the lack of active mRNA destabilization and the continuation of
crhC
transcription, at least transiently, after a temperature upshift. Translational inhibitors do not induce
crhC
expression to cold shock levels, indicating that inhibition of translation is only one of the signals required to activate the cold shock response in
Anabaena
. Limited amounts of protein synthesis are required for the cold shock-induced accumulation of
crhC
transcripts, as normal levels of accumulation occur in the presence of tetracycline but are abolished by chloramphenicol. Regulation of
crhC
expression may also extend to the translational level, as CrhC protein levels do not correlate completely with the pattern of mRNA transcript accumulation. Our experiments indicate that the regulation of
crhC
transcript accumulation is tightly controlled by both temperature and metabolic activity at the levels of transcription, mRNA stabilization, and translation.
Novel Missense Mutations That Affect the Transport Function of MalK, the ATP-Binding-Cassette Subunit of the
Salmonella enterica
Serovar Typhimurium Maltose Transport System
Authors: Hunke Sabine , Landmesser Heidi , Schneider Erwin 
Published: July 2002

ABSTRACT

We report on novel mutations in the
malK
gene of
Salmonella enterica
serovar Typhimurium, encoding the ATPase subunit of the maltose transporter (MalFGK
2
). Biochemical analysis suggests that (i) L86 might be involved in a signaling step during substrate translocation and (ii) E306 may be critical for the structural integrity of the protein.
ssrA
(tmRNA) Plays a Role in
Salmonella enterica
Serovar Typhimurium Pathogenesis
Authors: Julio Steven M. , Heithoff Douglas M. , Mahan Michael J. 
Published: July 2002

ABSTRACT

Escherichia coli ssrA
encodes a small stable RNA molecule, tmRNA, that has many diverse functions, including tagging abnormal proteins for degradation, supporting phage growth, and modulating the activity of DNA binding proteins. Here we show that
ssrA
plays a role in
Salmonella enterica
serovar Typhimurium pathogenesis and in the expression of several genes known to be induced during infection. Moreover, the phage-like attachment site,
attL
, encoded within
ssrA
, serves as the site of integration of a region of
Salmonella
-specific sequence; adjacent to the 5′ end of
ssrA
is another region of
Salmonella
-specific sequence with extensive homology to predicted proteins encoded within the unlinked
Salmonella
pathogenicity island SPI4.
S. enterica
serovar Typhimurium
ssrA
mutants fail to support the growth of phage P22 and are delayed in their ability to form viable phage particles following induction of a phage P22 lysogen. These data indicate that
ssrA
plays a role in the pathogenesis of
Salmonella
, serves as an attachment site for
Salmonella
-specific sequences, and is required for the growth of phage P22.
A New IS
4
Family Insertion Sequence, IS
4Bsu
1, Responsible for Genetic Instability of Poly-γ-Glutamic Acid Production in
Bacillus subtilis
Authors: Nagai Toshiro , Phan Tran Lam-Son , Inatsu Yasuhiro , Itoh Yoshifumi 
Published: July 2002

ABSTRACT

Certain
Bacillus subtilis
strains, such as
B. subtilis
(
natto
) starter strains for the manufacture of natto (fermented soybeans), produce capsular poly-γ-glutamate (γPGA). In
B. subtilis
(
natto
), γPGA synthesis is controlled by the ComP-ComA two-component regulatory system and thereby induced at the beginning of the stationary growth phase. We have found a new insertion sequence (IS), designated IS
4Bsu1
, in the
comP
gene of a spontaneous γPGA-negative mutant of
B. subtilis
(
natto
) NAF4. IS
4Bsu1
(1,406 bp), the first IS discovered in
B. subtilis
, encodes a putative transposase (Tpase) with a predicted
M
r
of 34,895 (374 residues) which displays similarity to the Tpases of IS
4
family members. Southern blot analyses have identified 6 to 11 copies of IS
4Bsu1
, among which 6 copies were at the same loci, in the chromosomes of
B. subtilis
(
natto
) strains, including NAF4, three commercial starters, and another three γPGA-producing
B. subtilis
(
natto
) strains. All of the eight spontaneous γPGA
−
mutants, which were derived from five independent NAF4 cultures, had a new additional IS
4Bsu1
copy in
comP
at six different positions within 600 bp of the 5′-terminal region. The target sites of IS
4Bsu1
were determined to be AT-rich 9-bp sequences by sequencing the flanking regions of IS
4Bsu1
in mutant
comP
genes. These results indicate that IS
4Bsu1
transposes by the replicative mechanism, in contrast to other IS
4
members that use the conservative mechanism, and that most, if not all, of spontaneous γPGA
−
mutants appear to have resulted from the insertion of IS
4Bsu1
exclusively into
comP
. The presence of insertion hot spots in
comP
, which is essential for γPGA synthesis, as well as high transposition activity, would account for the high frequency of spontaneous γPGA
−
mutation by IS
4Bsu1
in
B. subtilis
(
natto
).
Renaturation of
Bacillus thermoglucosidasius
HrcA Repressor by DNA and Thermostability of the HrcA-DNA Complex In Vitro
Authors: Watanabe Kunihiko , Yamamoto Takeshi , Suzuki Yuzuru 
Published: July 2002

ABSTRACT

HrcA, a negative control repressor for chaperone expression from the obligate thermophile
Bacillus thermoglucosidasius
KP1006, was purified in a His-tagged form in the presence of 6 M urea but hardly renatured to an intact state due to extreme insolubility. Renaturation trials revealed that the addition of DNA to purified
B. thermoglucosidasius
HrcA can result in solubilization of HrcA free from the denaturing agent urea. Results from band shift and light scattering assays provided three new findings: (i) any species of DNA can serve to solubilize
B. thermoglucosidasius
HrcA, but DNA containing the CIRCE (controlling inverted repeat of chaperone expression) element is far more effective than other nonspecific DNA; (ii)
B. thermoglucosidasius
HrcA renatured with nonspecific DNA bound the CIRCE element in the molecular ratio of 2.6:1; and (iii)
B. thermoglucosidasius
HrcA binding to the CIRCE element was stable at below 50°C whereas the complex was rapidly denatured at 70°C, suggesting that the breakdown of HrcA is induced by heat stress and HrcA may act as a thermosensor to affect the expression of heat shock regulatory genes. These results will help to determine the nature of HrcA protein molecules.
Identification and Distribution of New Insertion Sequences in the Genome of Alkaliphilic
Bacillus halodurans
C-125
Authors: Takami Hideto , Han Chang-Gyun , Takaki Yoshihiro , Ohtsubo Eiichi 
Published: July 2002

ABSTRACT

Fifteen kinds of new insertion sequences (ISs), IS
641
to IS
643
, IS
650
to IS
658
, IS
660
, IS
662
, and IS
663
, and a group II intron (Bh.Int) were identified in the 4,202,352-bp genome of alkaliphilic
Bacillus halodurans
C-125. Out of 120 ISs identified in the C-125 genome, 29 were truncated, indicating the occurrence of internal rearrangements of the genome. The ISs other than IS
650
, IS
653
, IS
660
, and IS
663
generated a 2- to 9-bp duplication of the target site sequence, and the ISs other than IS
650
, IS
653
, and IS
657
carry 14- to 64-bp inverted repeats. Sequence analysis revealed that six kinds of ISs (IS
642
, IS
643
, IS
654
, IS
655
, IS
657
, and IS
658
) belong to a separate IS family (IS
630
, IS
21
, IS
256
, IS
3
, IS
200
/IS
605
, and IS
30
, respectively) as a new member. Also, IS
651
and IS
652
were characterized as new members of the IS
L3
family. Significant similarity was found between the transposase (Tpase) sequences between IS
650
and IS
653
(78.2%), IS
651
and IS
652
(56.3%), IS
656
and IS
662
(71.0%), and IS
660
and IS
663
(44.5%), but the others showed no similarity to one another. Tpases in 28 members of IS
651
in the C-125 genome were found to have become diversified. Most of the IS elements widely distributed throughout the genome were inserted in noncoding regions, although some genes, such as those coding for an ATP-binding cassette transporter/permease, a response regulator, and
l
-indole 2-dehydrogenase, have been mutated through the insertion of IS elements. It is evident, however, that not all IS elements have transposed and caused rearrangements of the genome in the past 17 years during which strain C-125 was subcultured under neutral and alkaline conditions.
Domain Interactions in the Yeast ATP Binding Cassette Transporter Ycf1p: Intragenic Suppressor Analysis of Mutations in the Nucleotide Binding Domains
Authors: Falcón-Pérez Juan M. , Martı́nez-Burgos Mónica , Molano Jesús , Mazón Marı́a J. , Eraso Pilar 
Published: July 2002

ABSTRACT

The yeast cadmium factor (Ycf1p) is a vacuolar ATP binding cassette (ABC) transporter required for heavy metal and drug detoxification. Cluster analysis shows that Ycf1p is strongly related to the human multidrug-associated protein (MRP1) and cystic fibrosis transmembrane conductance regulator and therefore may serve as an excellent model for the study of eukaryotic ABC transporter structure and function. Identifying intramolecular interactions in these transporters may help to elucidate energy transfer mechanisms during transport. To identify regions in Ycf1p that may interact to couple ATPase activity to substrate binding and/or movement across the membrane, we sought intragenic suppressors of
ycf1
mutations that affect highly conserved residues presumably involved in ATP binding and/or hydrolysis. Thirteen intragenic second-site suppressors were identified for the D777N mutation which affects the invariant Asp residue in the Walker B motif of the first nucleotide binding domain (NBD1). Two of the suppressor mutations (V543I and F565L) are located in the first transmembrane domain (TMD1), nine (A1003V, A1021T, A1021V, N1027D, Q1107R, G1207D, G1207S, S1212L, and W1225C) are found within TMD2, one (S674L) is in NBD1, and another one (R1415G) is in NBD2, indicating either physical proximity or functional interactions between NBD1 and the other three domains. The original D777N mutant protein exhibits a strong defect in the apparent affinity for ATP and
V
max
of transport. The phenotypic characterization of the suppressor mutants shows that suppression does not result from restoring these alterations but rather from a change in substrate specificity. We discuss the possible involvement of Asp777 in coupling ATPase activity to substrate binding and/or transport across the membrane.
Evidence for the Contribution of Point Mutations tovlsEVariation and for Apparent Constraints on the Net Accumulation of Sequence Changes invlsEduring Infection with Lyme Disease Spirochetes
Authors: Sung Shian-Ying , McDowell John V. , Marconi Richard T. 
Published: July 2002

ABSTRACTIn the Lyme disease spirochetes, both theospEandvlsEgene families have been demonstrated to undergo sequence variation during infection. To further investigate the mechanisms associated with the generation ofvlsvariation, single-nucleotide polymorphism and subsequent DNA sequence analyses were performed on thevlsEgene and its paralog, BBJ51, a related gene with a frameshift mutation. These analyses focused on a series of postinfection clonal populations obtained from mice infected withBorrelia burgdorferiB31MIpc or its clonal derivative, B31MIc53.vlsE, but not BBJ51, was found to undergo sequence changes during infection. Consistent with that reported previously (J.-R. Zhang et al., Cell 89:275–285, 1997) many of the sequence changes appear to have arisen through gene conversion events and to be localized to the variable regions ofvlsE. However, analysis of thevlsEnucleotide sequences revealed that some sequence changes were the result of point mutations, as these changes did not have potential contributing sources in thevlscassettes. To determine if sequence changes accumulate invlsEover long-term infection, thevlsEgenes of clonal populations recovered after 7 months of infection in mice were analyzed. While new sequence changes developed, a significant number of these changes resulted in the restoration of thevlsEsequence of the original infecting clone. In addition, we noted that some positions within the variable regions (VR) are stable even though the cassettes possess residues that could contribute to sequence variation through gene conversion. These analyses suggest that the total number of amino acid sequence changes that can be maintained by VlsE levels off during infection. In summary, in this report we demonstrate that the development of point mutations serves as a second mechanism by whichvlsEsequence variation can be generated and that the capacity forvlsEvariation, while still significant, is less than previously postulated.
Controlled Expression in
Klebsiella pneumoniae
and
Shigella flexneri
Using a Bacteriophage P1-Derived C1-Regulated Promoter System
Authors: Schofield David A. , Westwater Caroline , Dolan Joseph W. , Schmidt Michael G. , Norris James S. 
Published: July 2002

ABSTRACT

The utility of promoters regulated by the bacteriophage P1 temperature-sensitive C1 repressor was examined in
Shigella flexneri
and
Klebsiella pneumoniae
. Promoters carrying C1 operator sites driving LacZ expression had induction/repression ratios of up to 240-fold in
S. flexneri
and up to 50-fold in
K. pneumoniae
. The promoters exhibited remarkably low basal expression, demonstrated modulation by temperature, and showed rapid induction. This system will provide a new opportunity for controlled gene expression in enteric gram-negative bacteria.
Systematic Identification of Selective Essential Genes in
Helicobacter pylori
by Genome Prioritization and Allelic Replacement Mutagenesis
Authors: Chalker Alison F. , Minehart Heather W. , Hughes Nicky J. , Koretke Kristin K. , Lonetto Michael A. , Brinkman Kerry K. , Warren Patrick V. , Lupas Andrei , Stanhope Michael J. , Brown James R. , Hoffman Paul S. 
Published: July 2002

ABSTRACT

A comparative genomic approach was used to identify
Helicobacter pylori
26695 open reading frames (ORFs) which are conserved in
H. pylori
J99 but highly diverged in other eubacteria. A survey of selected pathways of central intermediary metabolism was also carried out, and genes with a potentially selective role in
H. pylori
were identified. Forty-five ORFs identified in these two analyses were screened using a rapid vector-free allelic replacement mutagenesis technique, and 33 were shown to be essential in vitro. Notably, 13 ORFs gave essentiality results which are unexpected in view of their known or proposed functions, and phylogenetic analysis was used to investigate the annotation of 7 such ORFs which are highly diverged. We propose that the products of a number of these
H. pylori
-specific essential genes may be suitable targets for novel anti-
H. pylori
therapies.
Hydrogen Peroxide-Forming NADH Oxidase Belonging to the Peroxiredoxin Oxidoreductase Family: Existence and Physiological Role in Bacteria
Authors: Nishiyama Yoshitaka , Massey Vincent , Takeda Kouji , Kawasaki Shinji , Sato Junichi , Watanabe Toshihiro , Niimura Youichi 
Published: July 2002

ABSTRACT

Amphibacillus xylanus
and
Sporolactobacillus inulinus
NADH oxidases belonging to the peroxiredoxin oxidoreductase family show extremely high peroxide reductase activity for hydrogen peroxide and alkyl hydroperoxides in the presence of the small disulfide redox protein, AhpC (peroxiredoxin). In order to investigate the distribution of this enzyme system in bacteria, 15 bacterial strains were selected from typical aerobic, facultatively anaerobic, and anaerobic bacteria. AhpC-linked alkyl hydroperoxide reductase activities were detected in most of the tested strains, and especially high activities were shown in six bacterial species that grow well under aerobic conditions, including aerobic bacteria (
Alcaligenes faecalis
and
Bacillus licheniformis
) and facultatively anaerobic bacteria (
Amphibacillus xylanus
,
Sporolactobacillus inulinus
,
Escherichia coli
, and
Salmonella enterica
serovar Typhimurium). In the absence of AhpC, the purified enzymes from
A. xylanus
and
S. inulinus
catalyze the NADH-linked reduction of oxygen to hydrogen peroxide. Similar activities were observed in the cell extracts from each of these six strains. The cell extract of
B. licheniformis
revealed the highest AhpC-linked alkyl hydroperoxide reductase activity in the four strains, with
V
max
values for hydrogen peroxide and alkyl hydroperoxides being similar to those for the enzymes from
A. xylanus
and
S. inulinus
. Southern blot analysis of the three strains probed with the
A. xylanus
peroxiredoxin reductase gene revealed single strong bands, which are presumably derived from the individual peroxiredoxin reductase genes. Single bands were also revealed in other strains which show high AhpC-linked reductase activities, suggesting that the NADH oxidases belonging to the peroxiredoxin oxidoreductase family are widely distributed and possibly play an important role both in the peroxide-scavenging systems and in an effective regeneration system for NAD in aerobically growing bacteria.
Western Blot Analysis of Sera Reactive to Human Monocytic Ehrlichiosis and Human Granulocytic Ehrlichiosis Agents
Authors: Unver Ahmet , Felek Suleyman , Paddock Christopher D. , Zhi Ning , Horowitz Harold W. , Wormser Gary P. , Cullman Louis C. , Rikihisa Yasuko 
Published: July 2002

ABSTRACT

Laboratory diagnosis of human ehrlichioses is routinely made by an indirect immunofluorescence assay (IFA) using cultured ehrlichia-infected whole cells as antigen. Concern has been raised that incorrect diagnoses of human monocytic ehrlichiosis (HME) or human granulocytic ehrlichiosis (HGE) may be made on the basis of serologic cross-reactivity between
Ehrlichia chaffeensis
and the agent of HGE. The present study examined whether two recombinant major outer membrane proteins, rP30 and rP44, that were previously shown to be sensitive and specific serodiagnostic antigens for HME and HGE, respectively, could be used to discriminate IFA dually reacting sera. Thirteen dually IFA-reactive sera, three sera that were IFA positive only with
E. chaffeensis
, and three sera that were IFA positive only with the HGE agent were examined by Western immunoblot analysis using purified whole organisms and recombinant proteins as antigens. All 16
E. chaffeensis
IFA-positive sera reacted with rP30. However, none of these sera reacted with rP44, regardless of IFA reactivity with the HGE agent. The three HGE-agent-only IFA-positive sera reacted only with rP44, not with rP30. Western immunoblotting using purified
E. chaffeensis
and the HGE agent as antigens suggested that heat shock and other proteins, but not major outer membrane proteins, cross-react between the two organisms. Therefore, Western immunoblot analysis using rP44 and rP30 may be useful in discriminating dually HME and HGE IFA-reactive sera.