bluuebunny/crossref_metadata_2025_split huggingface dataset:
Peptidyl‐tRNA hydrolase in Bacillus subtilis, encoded by spoVC, is essential to vegetative growth, whereas the homologous enzyme in Saccharomyces cerevisiae is dispensable
Authors: Menez Jeanne , Buckingham Richard H. , Zamaroczy Miklos de , Campelli Celine Karmazyn 
Published: March 2003

SummaryPeptidyl‐tRNA hydrolase in Escherichia coli, encoded by pth, is essential for recycling tRNA molecules sequestered as peptidyl‐tRNA as a result of pre‐mature dissociation from the ribosome during translation. Genes homologous to pth are present in other bacteria, yeast and man, but not in archaea. The homologous gene in Bacillus subtilis, spoVC, was first identified as a gene involved in sporulation. A second copy of spoVC, under the control of the xyl promoter, was integrated into B. subtilis at the amy locus. In this background, interruption of the original gene was possible provided that expression of the copy at the amy locus was induced. When spoVC was interrupted, both vegetative growth and sporulation were dependent on xylose, showing that SpoVC is essential. The role of SpoVC in sporulation is discussed and appears to be consistent with previous hypotheses that a relaxation of translational accuracy may occur during sporulation. The homologous gene in Saccharomyces cerevisiae, yHR189W, has been interrupted in both haploid and diploid strains. The mutant haploid strains remain viable, as do the yHR189W mutant spores obtained by tetrad dis‐section, with either glucose or glycerol as carbon source, showing that the yHR189W gene product is dispensable for cell growth and for mitochondrial respiration.
Identification of MrpI as the sole recombinase that regulates the phase variation of MR/P fimbria, a bladder colonization factor of uropathogenic Proteus mirabilis
Authors: Li Xin , Lockatell C. Virginia , Johnson David E. , Mobley Harry L. T. 
Published: March 2003

Summary Proteus mirabilis
is a common cause of urinary tract infection (UTI) in individuals with structural abnormalities or long‐term catheterization. The expression of mannose‐resistant/
Proteus
‐like (MR/P) fimbria is phase variable because of the inversion of a 251 bp DNA fragment that carries the promoter for the
mrp
operon. Previous studies have shown that
mrpI
, which is transcribed divergently from the
mrp
operon, encodes a recombinase capable of switching the orientation of this invertible element. In this study, we constructed isogenic
mrpI
null mutants from a clinical isolate of
P. mirabilis
, HI4320. A polymerase chain reaction (PCR)‐based invertible element assay revealed that the isogenic
mrpI
null mutants were locked in one phase, either expressing (locked on) MR/P fimbriae or not (locked off), which indicated that MrpI was the sole recombinase that regulated the phase variation of MR/P fimbria. The locked‐on and locked‐off mutants were evaluated for virulence in the CBA mouse model of ascending UTI by co‐challenges with each other and with the wild‐type strain. Results from these experiments demonstrated conclusively that the MR/P fimbria was a critical bladder colonization factor of uropathogenic
P. mirabilis and also suggested that the ability to switch off the expression of MR/P fimbria might be important for kidney colonization.
Global analysis of the Bacillus subtilis Fur regulon and the iron starvation stimulon
Authors: Baichoo Noel , Wang Tao , Ye Rick , Helmann John D. 
Published: March 2003

SummaryThe Bacillus subtilis ferric uptake repressor (Fur) protein coordinates a global transcriptional response to iron starvation. We have used DNA microarrays to define the Fur regulon and the iron starvation stimulon. We identify 20 operons (containing 39 genes) that are derepressed both by mutation of fur and by treatment of cells with the iron chelator 2,2′‐dipyridyl. These operons are direct targets of Fur regulation as judged by DNase I footprinting. Analyses of lacZ reporter fusions to six Fur‐regulated promoter regions reveal that repression is highly selective for iron. In addition to the Fur regulon, iron starvation induces members of the PerR regulon and leads to reduced expression of cytochromes. However, we did not find any evidence for genes that are directly activated by Fur or repressed by Fur under iron‐limiting conditions. Although genome searches using the 19 bp Fur box consensus are useful in identifying candidate Fur‐regulated genes, some genes associated with Fur boxes are not demonstrably regulated by Fur, whereas other genes are regulated from sites with little apparent similarity to the conventional Fur consensus.
Transcriptome analysis of all two‐component regulatory system mutants of Escherichia coli K‐12
Authors: Oshima Taku , Aiba Hirofumi , Masuda Yasushi , Kanaya Shigehiko , Sugiura Masahito , Wanner Barry L. , Mori Hirotada , Mizuno Takeshi 
Published: March 2003

SummaryWe have systematically examined the mRNA profiles of 36 two‐component deletion mutants, which include all two‐component regulatory systems of Escherichia coli, under a single growth condition. DNA microarray results revealed that the mutants belong to one of three groups based on their gene expression profiles in Luria–Bertani broth under aerobic conditions: (i) those with no or little change; (ii) those with significant changes; and (iii) those with drastic changes. Under these conditions, the anaeroresponsive ArcB/ArcA system, the osmoresponsive EnvZ/OmpR system and the response regulator UvrY showed the most drastic changes. Cellular functions such as flagellar synthesis and expression of the RpoS regulon were affected by multiple two‐component systems. A high correlation coefficient of expression profile was found between several two‐component mutants. Together, these results support the view that a network of functional interactions, such as cross‐regulation, exists between different two‐component systems. The compiled data are avail‐able at our website (http:ecoli.aist‐nara.ac.jpxpanalysis 2components).
Structural heterogeneity of carbohydrate modifications affects serospecificity of Campylobacter flagellins
Authors: Logan Susan M. , Kelly John F. , Thibault Pierre , Ewing Cheryl P. , Guerry Patricia 
Published: March 2003

SummaryFlagellin from Campylobacter coli VC167 is post‐translationally modified at ≥ 16 amino acid residues with pseudaminic acid and three related derivatives. The predominant modification was 5,7‐diacetamido‐3,5,7,9 ‐ tetradeoxy ‐ l ‐ glycero ‐ l ‐ manno ‐ nonulosonic acid (pseudaminic acid, Pse5Ac7Ac), a modification that has been described previously on flagellin from Campylobacter jejuni 81‐176. VC167 lacked two modi‐fications present in 81‐176 and instead had two unique modifications of masses 431 and 432 Da. Flagellins from both C. jejuni 81‐176 and C. coli VC167 were also modified with an acetamidino form of pseudaminic acid (PseAm), but tandem mass spectrometry indicated that the structure of PseAm differed in the two strains. Synthesis of PseAm in C. coli VC167 requires a minimum of six ptm genes. In contrast, PseAm is synthesized in C. jejuni 81‐176 via an alternative pathway using the product of the pseA gene. Mutation of the ptm genes in C. coli VC167 can be detected by changes in apparent Mr of flagellin in SDS‐PAGE gels, changes in isoelectric focusing (IEF) patterns and loss of immunoreactivity with antiserum LAH2. These changes corresponded to loss of both 315 Da and 431 Da modifications from flagellin. Complementation of the VC167 ptm mutants with the 81‐176 pseA gene in trans resulted in flagellins containing both 315 and 431 Da modifications, but these flagellins remained unreactive in LAH2 antibody, suggesting that the unique form of PseAm encoded by the ptm genes contributes to the serospecificity of the flagellar filament.
Characterization of Exc, a novel protein required for the excision of Bacteroides conjugative transposon
Authors: Sutanto Yuri , Shoemaker Nadja B. , Gardner Jeffrey F. , Salyers Abigail A. 
Published: March 2003

SummaryConjugative transposons are integrated elements that excise from the chromosome, then transfer by conjugation to a recipient in which they integrate once again. Recently, a gene, designated exc, was shown to be essential for excision of the Bacteroides conjugative transposon (CTnDOT) from the chromosome. The deduced amino acid sequence of Exc had low amino acid sequence similarity to DNA topoisomerase III, an enzyme that relaxes DNA supercoils. This similarity raised the question of whether Exc protein was a topoisomerase and, if so, whether topoisomerase activity might contribute to the excision process. Here, we demonstrate that Exc does have topoisomerase activity in vitro. Exc relaxed supercoiled DNA, had a conserved tyrosine as its active site and required magnesium ions for its relaxation activity. However, although mutation of the catalytic tyrosine of Exc to phenylalanine abolished the ability of the enzyme to relax DNA supercoils in vitro, the mutation did not abolish the ability of the protein to mediate excision in vivo. This surprising result suggests that CTnDOT excision does not rely on the topoisomerase activity of Exc in vivo.
Strand‐specific loading of DnaB helicase by DnaA to a substrate mimicking unwound oriC
Authors: Weigel Christoph , Seitz Harald 
Published: March 2003

SummaryWe analysed the enzymatic activity (strand dis‐placement) of the Escherichia coli DnaB helicase on a mirror‐image pair of oligonucleotide‐based substrates mimicking the unwound replication origin oriC. Loading of the helicase complex occurred exclusively to the single‐stranded ‘lower strand’ part of the substrates. Full helicase activity required DnaA bound to the double‐stranded part of the substrates (oriC DnaA box R1) and to their single‐stranded ‘upper strand’ part. We assume that in vivo DnaA also loads the first of two helicase complexes – required for the assembly of two replication forks – to the lower strand of oriC during initiation of bidirectional chromosome replication in E. coli.
SycE allows secretion of YopE–DHFR hybrids by the Yersinia enterocolitica type III Ysc system
Authors: Feldman Mario F. , Müller Simone , Wüest Esther , Cornelis Guy R. 
Published: March 2003

SummaryThe Ysc type III secretion system allows Yersinia enterocolitica to translocate virulence proteins, called Yop effectors, into the cytosol of eukaryotic cells. Some of the Yop effectors possess an individual chaperone called a Syc protein. The first 15 amino acids of the YopE effector constitute a secretion signal that is sufficient to promote secretion of several reporter proteins. Residues 15–50 of YopE comprise the minimal binding domain for the SycE chaperone. In this study, we investigated the secretion by the Ysc system of several YopE–DHFR hybrid proteins with different folding properties, and evaluated the role of SycE, the cognate chaperone of YopE, in this context. We have analysed the secretion of hybrids containing 16 (YopE16), 52 (YopE52) and 80 (the complete region covered by the chaperone, YopE80) amino acids of YopE or full‐length YopE (YopEFL) with wild‐type DHFR and two mutants with altered folding properties. The hybrids containing DHFRΔ77, the mutant whose folding properties are the most drastically affected, could be secreted in all the conditions tested, even in the absence of the chaperone SycE. In contrast, DHFRwt could only be secreted fused to the first 52 amino acids of YopE, and its secretion was strictly dependent on SycE. The hybrids YopE80–DHFRwt and YopEFL–DHFRwt were not secreted. YopEFL–DHFRwt completely jammed the channel in an SycE‐dependent fashion. Our experiments indicate that, in order to be secreted, proteins must be unfolded or only partially folded, and that TSS chaperones could keep their substrates in a secretion‐competent conformation, probably by preventing their folding. In addition, they show that the secretion apparatus can reject folded proteins if they are not deeply engaged into the injectisome.
Distribution and function of new bacterial intein‐like protein domains
Authors: Amitai Gil , Belenkiy Olga , Dassa Bareket , Shainskaya Alla , Pietrokovski Shmuel 
Published: March 2003

SummaryHint protein domains appear in inteins and in the C‐terminal region of Hedgehog and Hedgehog‐like animal developmental proteins. Intein Hint domains are responsible and sufficient for protein‐splicing of their host‐protein flanks. In Hedgehog proteins the Hint domain autocatalyses its cleavage from the N‐terminal domain of the Hedgehog protein by attaching a cholesterol molecule to it. We identified two new types of Hint domains. Both types have active site sequence features of Hint domains but also possess distinguishing sequence features. The new domains appear in more than 50 different proteins from diverse bacteria, including pathogenic species of humans and plants, such as Neisseria meningitidis and Pseudomonas syringae. These new domains are termed bacterial intein‐like (BIL) domains. Bacterial intein‐like domains are present in variable protein regions and are typically flanked by domains that also appear in secreted proteins such as filamentous haemagglutinin and calcium binding RTX repeats. Phylogenetic and genomic analysis of BIL sequences suggests that they were positively selected for in different lineages. We cloned two BIL domains of different types and showed them to be active. One of the domains efficiently cleaved itself from its C‐terminal flank and could also protein‐splice its two flanks, in E. coli and in a cell free system. We discuss several possible biological roles for BIL domains including microevolution and post translational modification for generating protein variability.
Unravelling the biology of macrophage infection by gene expression profiling of intracellular Salmonella enterica
Authors: Eriksson Sofia , Lucchini Sacha , Thompson Arthur , Rhen Mikael , Hinton Jay C. D. 
Published: March 2003

SummaryFor intracellular pathogens such as Salmonellae, Mycobacteriae and Brucellae, infection requires adaptation to the intracellular environment of the phagocytic cell. The transition from extracellular to intravacuolar environment has been expected to involve a global modulation of bacterial gene expression, but the precise events have been difficult to determine. We now report the complete transcriptional profile of intracellular Salmonella enterica sv. Typhimurium following macrophage infection. During replication in murine macrophage‐like J774‐A.1 cells, 919 of 4451 S. Typhimurium genes showed significant changes in transcription. The expression profile identified alterations in numerous virulence and SOS response genes and revealed unexpected findings concerning the biology of the Salmonella–macrophage interaction. We observed that intracellular Salmonella are not starved for amino acids or iron (Fe2+), and that the intravacuolar environment is low in phosphate and magnesium but high in potassium. S. Typhimurium appears to be using the Entner–Douderoff pathway to use gluconate and related sugars as a carbon source within macrophages. Almost half the in vivo‐regulated genes were of unknown function, suggesting that intracellular growth involves novel macrophage‐associated functions. This is the first report that identifies the whole set of in vivo‐regulated genes for any bacterial pathogen during infection of mammalian cells.
Widespread functional specialization of Plasmodium falciparum erythrocyte membrane protein 1 family members to bind CD36 analysed across a parasite genome
Authors: Robinson Bridget A. , Welch Teresa L. , Smith Joseph D. 
Published: March 2003

Summary Plasmodium falciparum
‐infected erythrocytes sequester from blood circulation by binding host endothelium. A large family of variant proteins mediates cytoadherence and their binding specificity determines parasite sequestration patterns and potential for disease. The aim of the present study was to understand how binding properties are encoded into family members and to develop sequence algorithms for predicting binding. To accomplish these goals computational approaches and a binding assay were used to characterize adhesion across
Plasmodium falciparum erythrocyte membrane 1 (PfEMP1) proteins in the 3D7 parasite genome. We report that most family members encode the capacity to bind CD36 in the protein's semi‐conserved head structure and describe the sequence characteristics of a group of PfEMP1 proteins that do not. Structural and functional grouping of PfEMP1 proteins based upon head structure and additional domain architectural properties provide new insights into the protein family. These can be used to investigate the role of proteins in malaria pathogenesis and potentially tailor vaccines to recognize particular binding variants.
Demonstration of a sensory rhodopsin in eubacteria
Authors: Jung Kwang‐Hwan , Trivedi Vishwa D. , Spudich John L. 
Published: March 2003

SummaryWe report the first sensory rhodopsin observed in the eubacterial domain, a green light‐activated photoreceptor in Anabaena (Nostoc) sp. PCC7120, a freshwater cyanobacterium. The gene encoding the membrane opsin protein of 261 residues (26 kDa) and a smaller gene encoding a soluble protein of 125 residues (14 kDa) are under the same promoter in a single operon. The opsin expressed heterologously in Escherichia coli membranes bound all‐trans retinal to form a pink pigment (λmax 543 nm) with a photochemical reaction cycle of 110 ms half‐life (pH 6.8, 18°C). Co‐expression with the 14 kDa protein increased the rate of the photocycle, indicating physical interaction with the membrane‐embedded rhodopsin, which we confirmed in vitro by affinity enrichment chromatography and Biacore interaction. The pigment lacks the proton donor carboxylate residue in helix C conserved in known retinylidene proton pumps and did not exhibit detectable proton ejection activity. We detected retinal binding to the protein in Anabaena membranes by SDS‐PAGE and autofluorography of 3H‐labelled all‐trans retinal of reduced membranes from the organism. We conclude that Anabaena rhodopsin functions as a photosensory receptor in its natural environment, and suggest that the soluble 14 kDa protein transduces a signal from the receptor. Therefore, unlike the archaeal sensory rhodopsins, which transmit signals by transmembrane helix–helix interactions with membrane‐embedded transducers, the Anabaena sensory rhodopsin may signal through a soluble cytoplasmic protein, analogous to higher animal visual pigments.
Effect of successive swallows on oesophageal motility of normal volunteers, patients with Chagas’ disease and patients with idiopathic achalasia
Authors: Dantas R. O. 
Published: March 2003

Abstract  Most frequently, ten swallows of a 5‐mL bolus of water are performed during oesophageal manometry. Our hypothesis is that five swallows may produce the same results. We studied the oesophageal contraction parameters of 40 volunteers, 75 patients with Chagas’ disease and 14 patients with idiopathic achalasia. Motility was recorded at 5, 10 and 15 cm above the lower oesophageal sphincter. The subjects performed ten swallows of a 5‐mL bolus of water alternated with ten dry swallows with an interval of at least 30 s. We measured the amplitude, duration, peristaltic velocity, number of failed and number of simultaneous contractions of the initial five and final five dry and wet swallows. The comparison of dry and wet swallows showed the differences already known. The comparison of the parameters of the initial five swallows with those of the final five swallows showed no differences. Thus, when the initial five or the final five swallows were considered, there was no change in the conclusions reached by the comparison of patients and volunteers and of dry and wet swallows. We conclude that five swallows may be sufficient for the manometric examination of oesophageal parameters in Chagas’ disease and idiopathic achalasia.
Effects of position on oesophageal function: studies using combined manometry and multichannel intraluminal impedance
Authors: Tutuian R. , Elton J. P. , Castell D. O. , Gideon R. Matthew , Castell J. A. , Katz P. O. 
Published: March 2003

Abstract Multichannel intraluminal impedance (MII) allows assessment of intraoesophageal bolus transit. In the supine position, bolus transit is produced almost exclusively by peristaltic contractions; in the upright position, gravity also contributes to bolus transit. MII and peristaltic pressures were measured in four positions (0, 30, 60 and 90°) using ten swallows (5 cc each) of both water and viscous liquid with body position determined by random choice. Tracings were analysed for total bolus transit time: time interval between bolus entry at 20 cm above and bolus exit at 5 cm above the lower oesophageal sphincter (LOS) and contraction amplitudes at 5 and 10 cm above the LOS. Statistical comparison of mean values of all four body positions was done using anova and Bonnferoni post‐test. Ten normal subjects (five females and five males, age 24–45 years) completed the study. At each body position, liquid material transited faster (P < 0.001) than viscous material. Both liquid and viscous materials transited at lower inclinations (0 and 30°) significantly slower than at higher inclinations (60 and 90°). There was an almost perfect inverse linear correlation between angle of inclination and bolus transit time for both liquid (r = −0.99) and viscous (r = −1.00) boluses (Spearman correlation r > 0.99 and P < 0.02 for both substances). Contraction amplitudes for liquid vs viscous material were not significantly different at a given degree of inclination. Mean distal oesophageal amplitude declined with increasing inclination. Combined MII–OM identifies and quantifies the effects of gravity on the dichotomy between specific pressures measured by OM and function assessed as transit measured by MII.
Analysis of the PTEN gene in human meningiomas
Authors: Peters  , Wellenreuther  , Rollbrocker  , Hayashi  , Meyer‐Puttlitz  , Duerr  , Lenartz  , Marsh  , Schramm  , Wiestler  , Parsons  , Eng  , Von Deimling  
Published: March 2003

N. Peters, R. Wellenreuther, B. Rollbrocker, Y. Hayashi, B. Meyer‐Puttlitz, E‐M. Duerr, D. Lenartz, D.J. Marsh, J. Schramm, O.D. Wiestler, R. Parsons, C. Eng & A. von Deimling (1998) Neuropathology and Applied Neurobiology24, 3–8Analysis of the PTEN gene in human meningiomasPrevious observations demonstrated that the neurofibromatosis type 2 gene (NF2) plays an important role in the pathogenesis of the transitional, fibroblastic and malignant variants of human meningiomas. No specific genes have been associated with the pathogenesis of meningothelial meningiomas and with the progression to anaplastic meningiomas. However, allelic losses on chromosomal arms 1p, 10q and 14q have been implicated in the process of malignant progression. Recently, PTEN (phosphatase and tensin homolog deleted on chromosome ten) also termed MMAC1 (mutated in multiple advanced cancers 1) or TEP1 (TGF—regulated and epithelial cell‐enriched phosphatase), emerged as a candidate gene on chromosome 10q23.3. Initial studies revealed mutations of PTEN in limited series of glioblastomas, breast, kidney and prostate carcinomas mainly as cell lines. In order to evaluate the involvement of PTEN in the development of meningiomas, we have analysed the entire coding sequence of the gene in a series of 55 meningiomas (WHO grade I), 10 atypical meningiomas (WHO grade II) and 10 anaplastic meningiomas (WHO grade III). No PTEN mutations were seen in the WHO grade I meningiomas. However, one of the anaplastic meningiomas carried a somatic mutation. In addition, all tumours were examined for the presence of homozygous deletions of PTEN but these were not detected in any of the meningiomas. Our data suggest that mutations in PTEN are not involved in the formation of low grade meningiomas, but may contribute to malignant progression in a fraction of anaplastic meningiomas.