bluuebunny/crossref_metadata_2025_split huggingface dataset:
The RNA degradosome and poly(A) polymerase of Escherichia coli are required in vivo for the degradation of small mRNA decay intermediates containing REP‐stabilizers
Authors: Khemici Vanessa , Carpousis Agamemnon J. 
Published: December 2004

SummaryIn Escherichia coli, REP‐stabilizers are structural elements in polycistronic messages that protect 5′‐proximal cistrons from 3′→5′ exonucleolytic degradation. The stabilization of a protected cistron can be an important determinant in the level of gene expression. Our results suggest that RNase E, an endoribonuclease, initiates the degradation of REP‐stabilized mRNA. However, subsequent degradation of mRNA fragments containing a REP‐stabilizer poses a special challenge to the mRNA degradation machinery. Two enzymes, the DEAD‐box RNA helicase, RhlB and poly(A) polymerase (PAP) are required to facilitate the degradation of REP‐stabilizers by polynucleotide phosphorylase (PNPase). This is the first in vivo evidence that these enzymes are required for the degradation of REP‐stabilizers. Furthermore, our results show that REP degradation by RhlB and PNPase requires their association with RNase E as components of the RNA degradosome, thus providing the first in vivo evidence that this ribonucleolytic multienzyme complex is involved in the degradation of structured mRNA fragments.
A novel integrative and conjugative element (ICE) of Escherichia coli: the putative progenitor of the Yersinia high‐pathogenicity island
Authors: Schubert Sören , Dufke Severin , Sorsa Johanna , Heesemann Jürgen 
Published: December 2004

SummaryDiversification of bacterial species and pathotypes is largely caused by horizontal transfer of diverse DNA elements such as plasmids, phages and genomic islands (e.g. pathogenicity islands, PAIs). A PAI called high‐pathogenicity island (HPI) carrying genes involved in siderophore‐mediated iron acquisition (yersiniabactin system) has previously been identified in Yersinia pestis, Y. pseudotuberculosis and Y. enterocolitica IB strains, and has been characterized as an essential virulence factor in these species. Strikingly, an orthologous HPI is a widely distributed virulence determinant among Escherichia coli and other Enterobacteriaceae which cause extraintestinal infections. Here we report on the HPI of E. coli strain ECOR31 which is distinct from all other HPIs described to date because the ECOR31 HPI comprises an additional 35 kb fragment at the right border compared to the HPI of other E. coli and Yersinia species. This part encodes for both a functional mating pair formation system and a DNA‐processing region related to plasmid CloDF13 of Enterobacter cloacae. Upon induction of the P4‐like integrase, the entire HPI of ECOR31 is precisely excised and circularised. The HPI of ECOR31 presented here resembles integrative and conjugative elements termed ICE. It may represent the progenitor of the HPI found in Y. pestis and E. coli, revealing a missing link in the horizontal transfer of an element that contributes to microbial pathogenicity upon acquisition.
A HU‐like gene mutation in Rhizobium leguminosarum bv. viciae affects the expression of nodulation genes
Authors: Li Qiang , Feng Jie , Hu Hai‐Liang , Chen Xiao‐Chun , Li Feng‐Qing , Hong Guo‐Fan 
Published: December 2004

SummaryNodD is the major regulator of nod genes expression in rhizobia. Previously, a HU‐like protein in Rhizobium leguminosarum bv. viciae has been identified to bind specifically with nod promoters and be involved in in vitro nodD transcription, but its in vivo function remained unknown. In this work we have cloned and sequenced the R. leguminosarum bv. viciae gene, named hurL, for this HU‐like protein. Using the E. coli‐expressed HurL proteins, we proved that HurL had high affinity to several nod promoters and showed a stimulation effect on in vitro nodD transcription at appropriate concentration. The R. leguminosarum bv. viciae hurL gene was mutated by insertion of a kanamycin resistance cassette. The obtained hurL mutant strain M704 exhibited poor growth under free‐living conditions and failed to induce nodules on Pisum sativum cv. Frisson and Vicia hirsuta. Further studies of NodD production and nod genes‐lacZ fusions expression in the hurL mutant revealed that inactivation of hurL led to severe impairment in the nodD expression, repression in the inducible expression of nodA and nodF, and slight enhancement in the expression of px2, a gene identified earlier in this lab. These results suggested that hurL might be required for maintaining the normal expression of nod genes in R. leguminosarum bv. viciae.
R174 of Escherichia coli FtsZ is involved in membrane interaction and protofilament bundling, and is essential for cell division
Authors: Koppelman Cecile‐Marie , Aarsman Mirjam E. G. , Postmus Jarne , Pas Evelien , Muijsers Anton O. , Scheffers Dirk‐Jan , Nanninga Nanne , Den Blaauwen Tanneke 
Published: December 2004

SummaryWe investigated the interaction between FtsZ and the cytoplasmic membrane using inside‐out vesicles. Comparison of the trypsin accessibility of purified FtsZ and cytoplasmic membrane‐bound FtsZ revealed that the protruding loop between helix 6 and helix 7 is protected from trypsin digestion in the latter. This hydrophobic loop contains an arginine residue at position 174. To investigate the role of R174, this residue was replaced by an aspartic acid, and FtsZ‐R174D was fused to green fluorescent protein (GFP). FtsZ‐R174D‐GFP could localize in an FtsZ and in an FtsZ84(Ts) background at both the permissive and the non‐permissive temperature, and it had a reduced affinity for the cytoplasmic membrane compared with wild‐type FtsZ. FtsZ‐R174D could also localize in an FtsZ depletion strain. However, in contrast to wild‐type FtsZ, FtsZ‐R174D was not able to complement the ftsZ84 mutation or the depletion strain and induced filamentation. In vitro polymerization experiments showed that FtsZ‐R174D is able to polymerize, but that these polymers cannot form bundles in the presence of 10 mM CaCl2. This is the first description of an FtsZ mutant that has reduced affinity for the cytoplasmic membrane and does not support cell division, but is still able to localize. The mutant is able to form protofilaments in vitro but fails to bundle. It suggests that neither membrane interaction nor bundling is a requirement for initiation of cell division.
The high‐resolution functional map of bacteriophage SPP1 portal protein
Authors: Isidro Anabela , Santos Mário A. , Henriques Adriano O. , Tavares Paulo 
Published: December 2004

SummaryAn essential component in the assembly of nucleocapsids of tailed bacteriophages and of herpes viruses is the portal protein that is located at the unique vertex of the icosahedral capsid through which DNA movements occur. A library of mutations in the bacteriophage SPP1 portal protein (gp6) was generated by random mutagenesis of gene 6. Screening of the library allowed identification of 67 single amino acid substitutions that impair portal protein function. Most of the mutations cluster within stretches of a few amino acids in the gp6 carboxyl‐terminus. The mutations were divided into five classes according to the step of virus assembly that they impair: (1) production of stable gp6; (2) interaction of gp6 with the minor capsid protein gp7; (3) incorporation of gp6 in the procapsid structure; (4) DNA packaging; and (5) sizing of the packaged DNA molecule. Most of the mutations fell in classes 3 and 4. This is the first high‐resolution functional map of a portal protein, in which its function at different steps of viral assembly can be directly correlated with specific regions of its sequence. The work provides a framework for the understanding of central processes in the assembly of viruses that use specialized portals to govern entry and exit of DNA from the viral capsid.
Deletion analyses of the peptidoglycan‐associated lipoprotein Pal reveals three independent binding sequences including a TolA box
Authors: Cascales Eric , Lloubès Roland 
Published: December 2004

SummaryThe Tol–Pal system of the Escherichia coli cell envelope is composed of five proteins. TolQ, TolR and TolA form a complex in the inner membrane, whereas TolB is a periplasmic protein interacting with Pal, the peptidoglycan‐associated lipoprotein anchored to the outer membrane. This system is required for outer membrane integrity and has been shown to form a trans‐envelope bridge linking inner and outer membranes. The TolA–Pal interaction plays an important role in the function of this system and has been found to depend on the proton motive force and the TolQ and TolR proteins. The Pal lipoprotein interacts with many components, such as TolA, TolB, OmpA, the major lipoprotein and the murein layer. In this study, six pal deletions were constructed. The analyses of the resulting Pal protein functions and interactions defined an N‐terminal region of 40 residues, which can be deleted without any cell‐damaging effect, and three independent regions required for its interaction with TolA, OmpA and TolB or the peptidoglycan. The analyses of the integrity of the cells producing the various Pal lipoproteins revealed strong outer membrane destabilization only when binding regions were deleted. Furthermore, a conserved polypeptide sequence located downstream of the peptidoglycan binding motif of Pal was required for the TolA–Pal interaction and for the maintenance of outer membrane stability.
A search for amino acid substitutions that universally activate response regulators
Authors: Smith Jenny G. , Latiolais Jamie A. , Guanga Gerald P. , Pennington J. Daniel , Silversmith Ruth E. , Bourret Robert B. 
Published: December 2004

SummaryTwo‐component regulatory systems, typically composed of a sensor kinase to detect a stimulus and a response regulator to execute a response, are widely used by microorganisms for signal transduction. Response regulators exhibit a high degree of structural similarity and undergo analogous activating conformational changes upon phosphorylation. The activity of particular response regulators can be increased by specific amino acid substitutions, which either prolong the lifetime or mimic key features of the phosphorylated state. We probed the universality of response regulator activation by amino acid substitution. Thirty‐six mutations that activate 11 different response regulators were identified from the literature. To determine whether the activated phenotypes would be retained in the context of a different response regulator, we recreated 51 analogous amino acid substitutions at corresponding positions of CheY. About 55% of the tested substitutions completely or partially inactivated CheY, ≈ 30% were phenotypically silent, and ≈ 15% activated CheY. Three previously uncharacterized activated CheY mutants were found. The 94NS (and presumably 94NT) substitutions resulted in resistance to CheZ‐mediated dephosphorylation. The 113AP substitution led to enhanced autophosphorylation and may increase the fraction of non‐phosphorylated CheY molecules that populate the activated conformation. The locations of activating substitutions on the response regulator three‐dimensional structure are generally consistent with current understanding of the activation mechanism. The best candidates for potentially universal activating substitutions of response regulators identified in this study were 13DK and 113AP.
Signal quenching, detoxification and mineralization of vir gene‐inducing phenolics by the VirH2 protein of Agrobacterium tumefaciens
Authors: Brencic Anja , Eberhard Anatol , Winans Stephen C. 
Published: December 2004

SummaryPlant tumorigenesis by Agrobacterium tumefaciens requires approximately 20 Vir proteins, transcription of which is induced by a family of phenolic compounds released from plant wound sites. One Vir protein, VirH2, plays a role in the metabolism of at least one phenolic inducer inasmuch as it converts ferulic acid, a potent vir gene inducer, to the non‐inducer caffeate by O‐demethylation of a methoxyl group. Here, we tested VirH2‐dependent O‐demethylation of 16 other vir‐inducing phenolics, and detected this activity for each compound. However, O‐demethylation rates differed enormously, with the strongest vir gene inducers such as acetosyringone being demethylated extremely slowly. Compounds containing two methoxyl groups were demethylated at both positions. In general, phenolic inducers were more toxic than their demethylated counterparts. A virH2 mutant was more sensitive than the wild type to growth inhibition by virtually all phenolic inducers tested, indicating that VirH2 detoxifies these compounds. VirH2 also played a role in mineralization of some phenolics. It converted vanillate to protocatechuate, which was then mineralized via the β‐ketoadipate pathway. Vanillyl alcohol and vanillin were also mineralized after being oxidized to vanillate. All three compounds served as sole sources of carbon, whereas the remaining 13 compounds did not.
Analysis of HutP‐dependent transcription antitermination in the Bacillus subtilis hut operon: identification of HutP binding sites on hut antiterminator RNA and the involvement of the N‐terminus of HutP in binding of HutP to the antiterminator RNA
Authors: Oda Masanao , Kobayashi Nobuhiro , Fujita Masaya , Miyazaki Yuusuke , Sadaie Yoshito , Kurusu Yasurou , Nishikawa Satoshi 
Published: December 2004

SummaryWe investigated HutP‐dependent transcription antitermination of the Bacillus subtilis hut operon. In vitro transcription assays with the B. subtilisσA‐containing RNA polymerase indicated that HutP inhibits transcription termination at the internal terminator by binding to the antiterminator on hut mRNA in the presence of histidine. Ethylnitrosourea modification interference assays and mutational analyses of the interference sites showed that interaction of HutP with a region containing three UAG trinucleotide sequences, which is located on top of the antiterminator structure, is critical for hut antitermination in vivo. Results from kinetic analysis of binding of HutP to RNA containing various portions of the antiterminator sequences indicated that secondary structure is required for binding of HutP to the region containing three UAG triplets in the antiterminator. The in vivo HutP antiterminator activity was reduced by the mutations in the N‐terminal region of HutP. The HutP variants with H4A, R7A, I9A and Q26A mutations exhibited reduced binding affinities to the antiterminator RNA in vitro. A 25‐mer peptide consisting of amino acid residues 2–26 of HutP bound to the antiterminator RNA. These results indicated that the N‐terminus of HutP is involved in binding of HutP to the antiterminator RNA.
A Gα subunit controls zoospore motility and virulence in the potato late blight pathogen Phytophthora infestans
Authors: Latijnhouwers Maita , Ligterink Wilco , Vleeshouwers Vivianne G.A.A. , van West Pieter , Govers Francine 
Published: December 2004

SummaryThe heterotrimeric G‐protein pathway is a ubiquitous eukaryotic signalling module that is known to regulate growth and differentiation in many plant pathogens. We previously identified Pigpa1, a gene encoding a G‐protein α subunit from the potato late blight pathogen Phytophthora infestans. P. infestans belongs to the class oomycetes, a group of organisms in which signal transduction processes have not yet been studied at the molecular level. To elucidate the function of Pigpa1, PiGPA1‐deficient mutants were obtained by homology‐dependent gene silencing. The Pigpa1‐silenced mutants produced zoospores that turned six to eight times more frequently, causing them to swim only short distances compared with wild type. Attraction to the surface, a phenomenon known as negative geotaxis, was impaired in the mutant zoospores, as well as autoaggregation and chemotaxis towards glutamic and aspartic acid. Zoospore production was reduced by 20–45% in different Pigpa1‐silenced mutants. Transformants expressing constitutively active forms of PiGPA1, containing amino acid substitutions (R177H and Q203L), showed no obvious phenotypic differences from the wild‐type strain. Infection efficiencies on potato leaves ranged from 3% to 14% in the Pigpa1‐silenced mutants, compared with 77% in wild type, showing that virulence is severely impaired. The results prove that PiGPA1 is crucial for zoospore motility and for pathogenicity in an important oomycete plant pathogen.
Escherichia coliprereplication complex assembly is regulated by dynamic interplay among Fis, IHF and DnaA
Authors: Ryan Valorie T. , Grimwade Julia E. , Camara Johanna E. , Crooke Elliott , Leonard Alan C. 
Published: December 2004

SummaryInitiator DnaA and DNA bending proteins, Fis and IHF, comprise prereplication complexes (pre‐RC) that unwind theEscherichia colichromosome's origin of replication,oriC. Loss of either Fis or IHF perturbs synchronous initiation fromoriCcopies in rapidly growingE. coli. Based on dimethylsulphate (DMS) footprinting of purified proteins, we observed a dynamic interplay among Fis, IHF and DnaA on supercoiledoriCtemplates. Low levels of Fis inhibitedoriCunwinding by blocking both IHF and DnaA binding to low affinity sites. As the concentration of DnaA was increased, Fis repression was relieved and IHF rapidly redistributed DnaA to all unfilled binding sites onoriC. This behaviourin vitrois analogous to observed assembly of pre‐RC in synchronizedE. coli. We propose that as new DnaA is synthesized inE. coli, opposing activities of Fis and IHF ensure an abrupt transition from a repressed complex with unfilled weak affinity DnaA binding sites to a completely loaded unwound complex, increasing both the precision of DNA replication timing and initiation synchrony.
Identification of competence pheromone responsive genes in Streptococcus pneumoniae by use of DNA microarrays
Authors: Peterson Scott N. , Sung Chang Kyoo , Cline Robin , Desai Bhushan V. , Snesrud Erik C. , Luo Ping , Walling Jennifer , Li Haiying , Mintz Michelle , Tsegaye Getahun , Burr Patrick C. , Do Yu , Ahn Susie , Gilbert Joseph , Fleischmann Robert D. , Morrison Donald A. 
Published: December 2004

SummaryNatural genetic transformation in Streptococcus pneumoniae is controlled in part by a quorum‐sensing system mediated by a peptide pheromone called competence‐stimulating peptide (CSP), which acts to coordinate transient activation of genes required for competence. To characterize the transcriptional response and regulatory events occurring when cells are exposed to competence pheromone, we constructed DNA microarrays and analysed the temporal expression profiles of 1817 among the 2129 unique predicted open reading frames present in the S. pneumoniae TIGR4 genome (84%). After CSP stimulation, responsive genes exhibited four temporally distinct expression profiles: early, late and delayed gene induction, and gene repression. At least eight early genes participate in competence regulation including comX, which encodes an alternative sigma factor. Late genes were dependent on ComX for CSP‐induced expression, many playing important roles in transformation. Genes in the delayed class (third temporal wave) appear to be stress related. Genes repressed during the CSP response include ribosomal protein loci and other genes involved in protein synthesis. This study increased the number of identified CSP‐responsive genes from approximately 40 to 188. Given the relatively large number of induced genes (6% of the genome), it was of interest to determine which genes provide functions essential to transformation. Many of the induced loci were subjected to gene disruption mutagenesis, allowing us to establish that among 124 CSP‐inducible genes, 67 were individually dispensable for transformation, whereas 23 were required for transformation.
Differential transcriptome profiling identifies Plasmodium genes encoding pre‐erythrocytic stage‐specific proteins
Authors: Kaiser Karine , Matuschewski Kai , Camargo Nelly , Ross Jessica , Kappe Stefan H. I. 
Published: December 2004

SummaryInvasive sporozoite and merozoite stages of malaria parasites that infect mammals enter and subsequently reside in hepatocytes and red blood cells respectively. Each invasive stage may exhibit unique adaptations that allow it to interact with and survive in its distinct host cell environment, and these adaptations are likely to be controlled by differential gene expression. We used suppression subtractive hybridization (SSH) of Plasmodium yoelii salivary gland sporozoites versus merozoites to identify stage‐specific pre‐erythrocytic transcripts. Sequencing of the SSH library and matching the cDNA sequences to the P. yoelii genome yielded 25 redundantly tagged genes including the only two previously characterized sporozoite‐specific genes encoding the circumsporozoite protein (CSP) and thrombospondin‐related anonymous protein (TRAP). Twelve novel genes encode predicted proteins with signal peptides, indicating that they enter the secretory pathway of the sporozoite. We show that one novel protein bearing a thrombospondin type 1 repeat (TSR) exhibits an expression pattern that suggests localization in the sporozoite secretory rhoptry organelles. In addition, we identified a group of four genes encoding putative low‐molecular‐mass proteins. Two proteins in this group exhibit an expression pattern similar to TRAP, and thus possibly localize in the sporozoite secretory micronemes. Proteins encoded by the differentially expressed genes identified here probably mediate specific interactions of the sporozoite with the mosquito vector salivary glands or the mammalian host hepatocyte and are not used during merozoite–red blood cell interactions.
Smt3/SUMO and Ubc9 are required for efficient APC/C‐mediated proteolysis in budding yeast
Authors: Dieckhoff Patrick , Bolte Melanie , Sancak Yasemin , Braus Gerhard H. , Irniger Stefan 
Published: December 2004

SummaryUbiquitin‐mediated proteolysis triggered by the anaphase‐promoting complex/cyclosome (APC/C) is essential for sister chromatid separation and the mitotic exit. Like ubiquitylation, protein modification with the small ubiquitin‐related modifier SUMO appears to be important during mitosis, because yeast cells impaired in the SUMO‐conjugating enzyme Ubc9 were found to be blocked in mitosis and defective in cyclin degradation. Here, we analysed the role of SUMOylation in the metaphase/anaphase transition and in APC/C‐mediated proteolysis in Saccharomyces cerevisiae. We show that cells depleted of Ubc9 or Smt3, the yeast SUMO protein, mostly arrested with undivided nuclei and with high levels of securin Pds1. This metaphase block was partially relieved by a deletion of PDS1. The absence of Ubc9 or Smt3 also resulted in defects in chromosome segregation. Temperature‐sensitive ubc9‐2 mutants were delayed in proteolysis of Pds1 and of cyclin Clb2 during mitosis. The requirement of SUMOylation for APC/C‐mediated degradation was tested more directly in G1‐arrested cells. Both ubc9‐2 and smt3‐331 mutants were defective in efficient degradation of Pds1 and mitotic cyclins, whereas proteolysis of unstable proteins that are not APC/C substrates was unaffected. We conclude that SUMOylation is needed for efficient proteolysis mediated by APC/C in budding yeast.