bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Single, Specific Thymine Mutation in the ComK-Binding Site Severely Decreases Binding and Transcription Activation by the Competence Transcription Factor ComK of
Bacillus subtilis
Authors: Susanna Kim A. , Mironczuk Aleksandra M. , Smits Wiep Klaas , Hamoen Leendert W. , Kuipers Oscar P. 
Published: April 2007

ABSTRACT

The competence transcription factor ComK plays a central role in competence development in
Bacillus subtilis
by activating the transcription of the K regulon. ComK-activated genes are characterized by the presence of a specific sequence to which ComK binds, a K-box, in their upstream DNA region. Each K-box consists of two AT-boxes with the consensus sequence AAAA-(N)
5
-TTTT, which are separated by a flexible spacer resulting in either two, three, or four helical turns between the starting nucleotides of the repeating AT-box units. In this study, the effects of potential determinants of ComK regulation in K-boxes were investigated by testing ComK's transcription activation and DNA-binding affinity on altered K-boxes with mutations either in the spacer between the AT-boxes or in the consensus sequence of the AT-boxes. The most striking result demonstrates the importance of the second thymine base in the AT-boxes. Mutation of this T into a guanine resulted in a threefold reduction in transcription activation and DNA binding by ComK. Transcription activation, as well as DNA binding, was almost completely abolished when both AT-boxes contained a T
2
-to-G mutation. This result was corroborated by in silico analyses demonstrating that a combination of mutations at the T
2
positions of both AT-boxes is not found among any ComK-activated K-boxes, indicating that at least one consensus T
2
position is required to maintain a functional K-box. The results suggest an important structural role for T
2
in ComK binding, probably by its specific position in the minor groove of the DNA.
Flavodoxin:Quinone Reductase (FqrB): a Redox Partner of Pyruvate:Ferredoxin Oxidoreductase That Reversibly Couples Pyruvate Oxidation to NADPH Production in
Helicobacter pylori
and
Campylobacter jejuni
Authors: St. Maurice Martin , Cremades Nunilo , Croxen Matthew A. , Sisson Gary , Sancho Javier , Hoffman Paul S. 
Published: April 2007

ABSTRACT

Pyruvate-dependent reduction of NADP has been demonstrated in cell extracts of the human gastric pathogen
Helicobacter pylori.
However, NADP is not a substrate of purified pyruvate:ferredoxin oxidoreductase (PFOR), suggesting that other redox active enzymes mediate this reaction. Here we show that
fqrB
(HP1164), which is essential and highly conserved among the epsilonproteobacteria, exhibits NADPH oxidoreductase activity. FqrB was purified by nickel interaction chromatography following overexpression in
Escherichia coli
. The protein contained flavin adenine dinucleotide and exhibited NADPH quinone reductase activity with menadione or benzoquinone and weak activity with cytochrome
c
, molecular oxygen, and 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB). FqrB exhibited a ping-pong catalytic mechanism, a
k
cat
of 122 s
−1
, and an apparent

K
m

of 14 μM for menadione and 26 μM for NADPH. FqrB also reduced flavodoxin (FldA), the electron carrier of PFOR. In coupled enzyme assays with purified PFOR and FldA, FqrB reduced NADP in a pyruvate- and reduced coenzyme A (CoA)-dependent manner. Moreover, in the presence of NADPH, CO
2
, and acetyl-CoA, the PFOR:FldA:FqrB complex generated pyruvate via CO
2
fixation. PFOR was the rate-limiting enzyme in the complex, and nitazoxanide, a specific inhibitor of PFOR of
H. pylori
and
Campylobacter jejuni
, also inhibited NADP reduction in cell-free lysates. These capnophilic (CO
2
-requiring) organisms contain gaps in pathways of central metabolism that would benefit substantially from pyruvate formation via CO
2
fixation. Thus, FqrB provides a novel function in pyruvate metabolism and, together with production of superoxide anions via quinone reduction under high oxygen tensions, contributes to the unique microaerobic lifestyle that defines the epsilonproteobacterial group.
In Vivo-Selected Mutations in Methyl-Directed Mismatch Repair Suppress the Virulence Attenuation ofSalmonella damMutant Strains following Intraperitoneal, but Not Oral, Infection of Naïve Mice
Authors: Heithoff Douglas M. , Badie Golnaz , Julio Steven M. , Enioutina Elena Y. , Daynes Raymond A. , Sinsheimer Robert L. , Mahan Michael J. 
Published: April 2007

ABSTRACTSalmonella entericaserovar Typhimurium that lacks the DNA adenine methylase (Dam) ectopically expresses multiple genes that are preferentially expressed during infection, is attenuated for virulence, and confers heightened immunity in vaccinated hosts. The safety ofdammutantSalmonellavaccines was evaluated by screening within infected mice for isolates that have an increased capacity to cause disease relative to the attenuated parental strain. Sincedammutant strains are sensitive to the DNA base analog 2-aminopurine (2-AP), we screened for 2-AP-resistant (2-APr) isolates in systemic tissues of mice infected withdammutantSalmonella. Such 2-APrderivatives were isolated following intraperitoneal but not oral administration and were shown to be competent for infectivity via intraperitoneal but not oral infection of naïve mice. These 2-APrderivatives were deficient in methyl-directed mismatch repair and were resistant to nitric oxide, yet they retained the bile-sensitive phenotype of the parentaldammutant strain. Additionally, introduction of amutHnull mutation intodammutant cells suppressed the inherent defects in intraperitoneal infectivity and nitric oxide resistance, as well as overexpression of SpvB, an actin cytotoxin required forSalmonellasystemic survival. These data suggest that restoration of intraperitoneal virulence ofdammutant strains is associated with deficiencies in methyl-directed mismatch repair that correlate with the production of systemically related virulence functions.
Diffusible Signal Factor-Dependent Cell-Cell Signaling and Virulence in the Nosocomial PathogenStenotrophomonas maltophilia
Authors: Fouhy Yvonne , Scanlon Karl , Schouest Katherine , Spillane Charles , Crossman Lisa , Avison Matthew B. , Ryan Robert P. , Dow J. Maxwell 
Published: April 2007

ABSTRACTThe genome ofStenotrophomonas maltophiliaencodes a cell-cell signaling system that is highly related to the diffusible signal factor (DSF)-dependent system of the phytopathogenXanthomonas campestris. Here we show that inS. maltophilia, DSF signaling controls factors contributing to the virulence and antibiotic resistance of this important nosocomial pathogen.
Genomic SELEX Search for Target Promoters under the Control of the PhoQP-RstBA Signal Relay Cascade
Authors: Ogasawara Hiroshi , Hasegawa Akiko , Kanda Emi , Miki Takenori , Yamamoto Kaneyoshi , Ishihama Akira 
Published: April 2007

ABSTRACTRstBA, a two-component regulatory system ofEscherichia coliwith an unidentified regulatory function, is under the control of a Mg2+-sensing PhoQP two-component system. In order to identify the network of transcription regulation downstream of RstBA, we isolated a set of RstA-binding sequences from theE. coligenome by using the genomic SELEX system. A gel mobility shift assay indicated the binding of RstA to two SELEX DNA fragments, one including the promoter region ofasr(acid shock RNA) and another including the promoter forcsgD(a regulator of the curli operon). Using a DNase I footprinting assay, we determined the RstA-binding sites (RstA boxes) with the consensus sequence TACATNTNGTTACA. Transcription of theasrgene was induced 10- to 60-fold either in low-pH (pH 4.5) LB medium or in low-phosphate minimal medium as detected by promoter assay. The acid-induced in vivo transcription ofasrwas reduced after the deletion ofrstA. In vivo transcription of theasrpromoter was observed only in the presence of RstA. In agreement with the PhoQP-RstBA network, the addition of Mg2+led to a severe reduction of theasrpromoter activity, and the disruption ofphoPalso reduced theasrpromoter activity, albeit to a lesser extent. These observations altogether indicate that RstA is an activator ofasrtranscription. In contrast, transcription ofcsgDwas repressed by overexpression of RstA, indicating that RstA is a repressor forcsgD. With these data taken together, we conclude that the expression of bothasrandcsgDis under the direct control of the PhoQP-RstBA signal relay cascade.
Clocking Out: Modeling Phage-Induced Lysis of
Escherichia coli
Authors: Ryan Gillian L. , Rutenberg Andrew D. 
Published: April 2007

ABSTRACT

Phage λ lyses the host
Escherichia coli
at a precisely scheduled time after induction. Lysis timing is determined by the action of phage holins, which are small proteins that induce hole formation in the bacterium's cytoplasmic membrane. We present a two-stage nucleation model of lysis timing, with the nucleation of condensed holin rafts on the inner membrane followed by the nucleation of a hole within those rafts. The nucleation of holin rafts accounts for most of the delay of lysis after induction. Our simulations of this model recover the accurate lysis timing seen experimentally and show that the timing accuracy is optimal. An enhanced holin-holin interaction is needed in our model to recover experimental lysis delays after the application of membrane poison, and such early triggering of lysis is possible only after the inner membrane is supersaturated with holin. Antiholin reduces the delay between membrane depolarization and lysis and leads to an earlier time after which triggered lysis is possible.
Specific Bonds between an Iron Oxide Surface and Outer Membrane Cytochromes MtrC and OmcA from
Shewanella oneidensis
MR-1
Authors: Lower Brian H. , Shi Liang , Yongsunthon Ruchirej , Droubay Timothy C. , McCready David E. , Lower Steven K. 
Published: April 2007

ABSTRACT

Shewanella oneidensis
MR-1 is purported to express outer membrane cytochromes (e.g., MtrC and OmcA) that transfer electrons directly to Fe(III) in a mineral during anaerobic respiration. A prerequisite for this type of reaction would be the formation of a stable bond between a cytochrome and an iron oxide surface. Atomic force microscopy (AFM) was used to detect whether a specific bond forms between a hematite (Fe
2
O
3
) thin film, created with oxygen plasma-assisted molecular beam epitaxy, and recombinant MtrC or OmcA molecules coupled to gold substrates. Force spectra displayed a unique force signature indicative of a specific bond between each cytochrome and the hematite surface. The strength of the OmcA-hematite bond was approximately twice that of the MtrC-hematite bond, but direct binding to hematite was twice as favorable for MtrC. Reversible folding/unfolding reactions were observed for mechanically denatured MtrC molecules bound to hematite. The force measurements for the hematite-cytochrome pairs were compared to spectra collected for an iron oxide and
S. oneidensis
under anaerobic conditions. There is a strong correlation between the whole-cell and pure-protein force spectra, suggesting that the unique binding attributes of each cytochrome complement one another and allow both MtrC and OmcA to play a prominent role in the transfer of electrons to Fe(III) in minerals. Finally, by comparing the magnitudes of binding force for the whole-cell versus pure-protein data, we were able to estimate that a single bacterium of
S. oneidensis
(2 by 0.5 μm) expresses ∼10
4
cytochromes on its outer surface.
YbxF, a Protein Associated with Exponential-Phase Ribosomes inBacillus subtilis
Authors: Sojka Luděk , Fučík Vladimír , Krásný Libor , Barvík Ivan , Jonák Jiří 
Published: April 2007

ABSTRACTTheybxFgene is a member of the streptomycin operon in a wide range of gram-positive bacteria. InBacillus subtilis, it codes for a small basic protein (82 amino acids, pI 9.51) of unknown function. We demonstrate that, inB. subtilis, YbxF localizes to the ribosome, primarily to the 50S subunit, with dependence on growth phase. Based on three-dimensional structures of YbxF generated by homology modeling, we identified helix 2 as important for the interaction with the ribosome. Subsequent mutational analysis of helix 2 revealed Lys24 as crucial for the interaction. Neither theB. subtilis ybxFgene nor its paralogue, theymxCgene, is essential, as shown by probing ΔybxF, ΔymxC, or ΔybxFΔymxCdouble deletion strains in several functional assays.
l
-Sorbose Reductase and Its Transcriptional Regulator Involved in
l
-Sorbose Utilization of
Gluconobacter frateurii
Authors: Soemphol Wichai , Toyama Hirohide , Moonmangmee Duangtip , Adachi Osao , Matsushita Kazunobu 
Published: April 2007

ABSTRACT

Upstream of the gene for flavin adenine dinucleotide (FAD)-dependent
d
-sorbitol dehydrogenase (SLDH),
sldSLC
, a putative transcriptional regulator was found in
Gluconobacter frateurii
THD32 (NBRC 101656). In this study, the whole
sboR
gene and the adjacent gene,
sboA
, were cloned and analyzed.
sboR
mutation did not affect FAD-SLDH activity in the membrane fractions. The SboA enzyme expressed and purified from an
Escherichia coli
transformant showed NADPH-dependent
l
-sorbose reductase (NADPH-SR) activity, and the enzyme was different from the NADPH-SR previously reported for
Gluconobacter suboxydans
IFO 3291 in molecular size and amino acid sequence. A mutant defective in
sboA
showed significantly reduced growth on
l
-sorbose, indicating that the SboA enzyme is required for efficient growth on
l
-sorbose. The
sboR
mutant grew on
l
-sorbose even better than the wild-type strain did, and higher NADPH-SR activity was detected in cytoplasm fractions. Reverse transcription-PCR experiments indicated that
sboRA
comprises an operon. These data suggest that
sboR
is involved in the repression of
sboA
, but not in the induction of
sldSLC
, on
d
-sorbitol and that another activator is required for the induction of these genes by
d
-sorbitol or
l
-sorbose.
Genetic Diversity of Toxigenic and Nontoxigenic
Vibrio cholerae
Serogroups O1 and O139 Revealed by Array-Based Comparative Genomic Hybridization
Authors: Pang Bo , Yan Meiying , Cui Zhigang , Ye Xiaofen , Diao Baowei , Ren Yonghong , Gao Shouyi , Zhang Liang , Kan Biao 
Published: April 2007

ABSTRACT

Toxigenic serogroups O1 and O139 of
Vibrio cholerae
may cause cholera epidemics or pandemics. Nontoxigenic strains within these serogroups also exist in the environment, and also some may cause sporadic cases of disease. Herein, we investigate the genomic diversity among toxigenic and nontoxigenic O1 and O139 strains by comparative genomic microarray hybridization with the genome of El Tor strain N16961 as a base. Conservation of the toxigenic O1 El Tor and O139 strains is found as previously reported, whereas accumulation of genome changes was documented in toxigenic El Tor strains isolated within the 40 years of the seventh pandemic. High phylogenetic diversity in nontoxigenic O1 and O139 strains is observed, and most of the genes absent from nontoxigenic strains are clustered together in the N16961 genome. By comparing these toxigenic and nontoxigenic strains, we observed that the small chromosome of
V. cholerae
is quite conservative and stable, outside of the superintegron region. In contrast to the general stability of the genome, the superintegron demonstrates pronounced divergence among toxigenic and nontoxigenic strains. Additionally, sequence variation in virulence-related genes is found in nontoxigenic El Tor strains, and we speculate that these intermediate strains may have pathogenic potential should they acquire CTX prophage alleles and other gene clusters. This genome-wide comparison of toxigenic and nontoxigenic
V. cholerae
strains may promote understanding of clonal differentiation of
V. cholerae
and contribute to an understanding of the origins and clonal selection of epidemic strains.
Expression of ZNF652, a novel zinc finger protein, in vulvar carcinomas and its relation to prognosis
Authors: Holm R , Knopp S , Kumar R , Lee J , Nesland J M , Tropè C , Callen D F 
Published: April 2007

Aims:To determine the levels of expression of ZNF652 and its relevance to prognosis in vulvar squamous cell carcinomas.Methods:22 cases of vulvar intraepithelial neoplasia (VIN) and tumours from 217 patients with vulvar squamous cell carcinomas were investigated for expression of ZNF652 using immunostaining methods. The effect of ZNF652 ectopic expression was determined in the vulvar carcinoma cell line SW954 by western and cell-based assays.Results:High levels of ZNF652 nuclear expression were observed in 5 (100%) of VIN I, 6 (75%) of VIN II and 109 (50.2%) of the vulvar carcinomas, whereas low levels were seen in 2 (25%) VIN II, 9 (100%) of VIN III and 108 (49.8%) of the vulvar carcinomas. High levels of ZNF652 expression in the vulvar carcinomas were significantly correlated to high expression of EphA2. However, when correcting for multiple testing this correlation was lost. No association was identified between ZNF652 expression and p16, p21, p27, p53, cyclin A, D1, D3, E, EphrinA-1 and human papillomavirus. Variations in levels of ZNF652 were not related to prognosis. Low levels of ZNF652 protein were identified in the vulvar carcinoma cell line SW954. Furthermore, SW954 cells ectopically expressing ZNF652 showed reduced cell proliferation and the ability to form colonies on plastic.Conclusions:ZNF652 protein expression is reduced in 25% of VIN II, 100% of VIN III and approximately 50% of the cases of vulvar squamous cell carcinoma, and may be an early event in the pathogenesis of vulvar squamous cell carcinoma. Variations in the levels of ZNF652 were not related to patient’s prognosis.
Effects of fixation on RNA integrity in a liquid-based cervical cytology setting
Authors: Horvath C , Boulet G , Sahebali S , Bogers J , Depuydt C , Vereecken A , Vermeulen T , Vanden Broeck D 
Published: April 2007

Aims:Despite many improvements, cervical cancer screening is still subject to shortcomings. Diagnostic accuracy may improve by using molecular biological techniques, requiring RNA of superior quality. This study determined the effect of SurePath fixation on RNA integrity to assess the suitability of clinical samples collected in this medium for RNA-based molecular assays.Methods:RNA isolation was performed on fresh and fixed HeLa cells and exfoliated cervical cells fixed in SurePath. The RNA integrity was evaluated by analysis of ribosomal RNA as an indicator of quality. The effect of SurePath preservation on PCR amplification was evaluated by real-time reverse transcriptase (RT)-PCR.Results:In contrast to unfixed cells, SurePath-fixed cells yielded less and severely degraded RNA, as shown by the absence of ribosomal RNA bands. RNA derived from SurePath-fixed cells showed poor real-time RT-PCR amplification characteristics, as evidenced by the absent correlation between threshold values and log cDNA concentration.Conclusions:Implementation of molecular biology in a clinical context is on the rise and may alleviate shortcomings in current screening and diagnostics. This study shows that SurePath fixation gives rise to highly fragmented RNA with insufficient quality for further reliable analysis by standard real-time RT-PCR applications. The increasing prominence of molecular screening stresses the importance of this finding, which must be considered in relation to choice of an appropriate liquid-based cytology system.
Axillary sentinel lymph node micrometastases with extracapsular extension: a distinct pattern of breast cancer metastasis?
Authors: Cserni G 
Published: April 2007

Aims:To examine the frequency of extracapsular extension (ECE) of sentinel lymph node (SLN) metastases in breast cancer according to metastasis size, and to characterise ECE in micometastases.Methods:If initially negative, SLNs were examined by step-sectioning and immunohistochemistry. Non-SLNs were not subjected to enhanced pathology. Positive axillary SLNs were analysed for metastasis size and the presence of ECE.Results:Of 885 successful SLN biopsy cases, 343 (39%) exhibited SLN involvement, and 115 (34%) displayed ECE. Of the latter, 107 underwent axillary dissection, and 63 (59%) of these demonstrated non-SLN metastases. The presence of ECE correlated with metastasis size (coefficient 0.92). Eight (10%) of the 84 micrometastatic SLN cases had ECE, and two of these were associated with non-SLN metastases. Only ECE and only the intraparenchymal nodal part of the micrometastasis were seen in some sections of five cases each. The primary tumours of the micrometastatic cases with ECE were non-high-grade and often of tubular type.Conclusions:The frequency of ECE increases with increasing nodal metastasis size. Minimal nodal metastases with ECE may represent a distinct pattern of nodal involvement with a predominant capsular and extracapsular, but only minimal or no nodal parenchymal component, predominantly seen in non-poorly differentiated and/or tubular carcinomas. This presentation of nodal metastasis can sometimes pose differential diagnostic problems, and should be distinguished from massive metastases presenting with ECE because it does not seem to be so commonly associated with non-SLN metastases or a massive metastatic load to the axilla as ECE of SLN metastases in general.
Parafibromin expression in breast cancer: a novel marker for prognostication?
Authors: Selvarajan S , Sii L-H , Lee A , Yip G , Bay B-H , Tan M-H , Teh B-T , Tan P-H 
Published: April 2007

Background:Parafibromin is a novel protein product ofHRPT2, a recently identified tumour suppressor gene. Mutations of theHRPT2gene are common in parathyroid carcinomas, and these exhibit reduced protein expression. Parafibromin expression in breast cancer has not been previously studied.Aims:To determine the distribution of parafibromin in breast cancer tissues, and correlate its expression with conventional pathological parameters.Methods:Tissue microarrays were constructed from archival paraffin embedded breast cancer samples. Sections cut from tissue microarray blocks were subjected to immunohistochemistry. Immunopositivity for parafibromin and intensity-percentage scores were derived by blinded evaluation. Findings were correlated with clinicopathological parameters.Results:163 breast cancers were assessed. Larger tumours were less likely to express parafibromin than smaller ones, with the association approaching statistical significance (p = 0.05). Staining intensity correlated inversely with tumour size (p = 0.016) and pathological stage (p = 0.008); as did parafibromin intensity-percentage score with pathological stage (p = 0.03), lymphovascular invasion (p = 0.03) and cerbB2 intensity-percentage score (p = 0.04).Conclusion:Parafibromin in breast cancer, as in parathyroid tumours, appears to have tumour suppressor functions, with loss of protein expression associated with adverse pathological parameters. These findings may indicate a potential role of parafibromin as a prognostic marker in breast cancer.
Thermal expansion: a fresh look at an old problem
Authors: Mohazzabi Pirooz , Behroozi Feredoon 
Published: May 2007

The phenomenon of thermal expansion is discussed from a basic perspective. Using a new approach, the well-known equation that relates the interatomic distance to the mean total energy per atom in a solid is obtained. Unlike the standard derivation, the new treatment is free from the computational inconsistencies and yields the correct expression for thermal expansion at any temperature. The standard derivation of the equation is outlined and its shortcomings are discussed. PACS Nos.: 65.40.–b, 65.40.De