bluuebunny/crossref_metadata_2025_split huggingface dataset:
Interaction surface of the Spo0A response regulator with the Spo0E phosphatase
Authors: Stephenson Sophie J. , Perego Marta 
Published: March 2003

SummarySpo0A~P is the essential response regulator and transcription factor for sporulation initiation in Bacillus subtilis. The phosphorylation level of Spo0A in the

cell is determined by the sensor kinase activity of the phosphorelay, donating phosphoryl

groups, and the antagonistic effects of dephosphorylation mediated by the Rap and

Spo0E families of phosphatases. In this study, spo0A mutations were generated

that encoded proteins less sensitive to the activity of Spo0E than the wild‐type

protein. The Spo0A substitutions N12K, P60S, L62P and F88L are surface exposed and

localize to the same face of the molecule as the active site and in its close proximity

on the β1–α1, β3–α3 and

β4–α4 loops. The corresponding surface in the Spo0F response regulator was shown previously to be involved in the interaction with the RapB phosphatase, as well as the KinA histidine kinase and the Spo0B phosphotransferase. Thus, residues occupying the same position (N12:Q12, F88:Y84) and the same loops in Spo0A or Spo0F are involved in the interaction with the structurally unrelated Spo0E and RapB phosphatases, respectively, in addition to kinases and phosphotransferase. The specificity in phosphatase target recognition must be the result of side‐chain variability within the response regulators and the interactions they promote. The residues involved in Spo0E interaction are identical in all Spo0A orthologues from spore‐forming Bacilli encoding Spo0E phosphatases.
Identification of XcpP domains that confer functionality and specificity to the Pseudomonas aeruginosa type II secretion apparatus
Authors: Gérard‐Vincent Manon , Robert Viviane , Ball Geneviève , Bleves Sophie , Michel Gérard P. F. , Lazdunski Andrée , Filloux Alain 
Published: March 2003

SummaryGram‐negative bacteria have evolved several types of secretion mechanisms to release proteins into the extracellular medium. One such mechanism, the type II secretory system, is a widely conserved two‐step process. The first step is the translocation of signal peptide‐bearing exoproteins across the inner membrane. The second step, the translocation across the outer membrane, involves the type II secretory apparatus or secreton. The secretons are made up of 12–15 proteins (Gsp) depending on the organism. Even though the systems are conserved, hetero‐logous secretion is mostly species restricted. Moreover, components of the secreton are not systematically exchangeable, especially with distantly related microorganisms. In closely related species, two components, the GspC and GspD (secretin) family members, confer specificity for substrate recognition and/or secreton assembly. We used Pseudomonas aeruginosa as a

model organism to determine which domains of XcpP (GspC member) are involved in specificity.

By constructing hybrids between XcpP and OutC, the Erwinia chrysanthemi homologue, we identified a region of 35 residues that was not exchangeable. We showed that this region might influence the stability of the XcpYZ secreton subcomplex. Remarkably, XcpP and OutC have domains, coiled‐coil and PDZ, respectively, which exhibit the same function but that are structurally different. Those two domains are exchangeable and we provided evidence that they are involved in the formation of homomultimeric complexes of XcpP.
Direct and indirect roles of CcpA in regulation of Bacillus subtilis Krebs cycle genes
Authors: Kim Hyun‐Jin , Roux Agnes , Sonenshein Abraham L. 
Published: March 2003

SummaryCarbon catabolite repression of the Bacillus subtilis citrate synthase (citZ) and aconitase (citB) genes, previously known to be regulated by CcpC, was shown to depend on CcpA as well. Transcription of the citZ gene was partially derepressed in ccpA and ccpC single mutants and fully derepressed in a ccpA ccpC double mutant. DNase I footprinting studies showed that CcpA binds to a catabolite‐responsive element (cre) site located at positions +80 to +97 with respect to the transcription start site, whereas CcpC binds at positions −14 to +6 and +16 to +36. Mutations in the citZ cre site greatly altered CcpA binding and repression. A ccpA null mutation also caused partial derepression of citB. Disruption of citrate synthase activity, however, suppressed the effect of the ccpA mutation, suggesting that increased citrate accumulation in a ccpA mutant partially inactivates CcpC and causes partial derepression of citB. Therefore, CcpA controls expression of Krebs cycle genes directly by regulating transcription of citZ and in‐directly by regulating availability of citrate, the inducer for CcpC.
Survival mechanisms for Streptomyces linear replicons after telomere damage
Authors: Qin Zhongjun , Cohen Stanley N. 
Published: March 2003

SummaryThe ability of linear replicons to propagate their DNA after telomere damage is essential for perpetuation of the genetic information they carry. We introduced deletions at specific locations within telomeres of streptomycete linear plasmids and investigated mechanisms that enable survival. Here, we report that rescue of such plasmids in Streptomyces lividans occurs by three distinct types of events: (i) repair of the damaged telomere by homologous recombination; (ii) circularization of the plasmid by non‐homologous end‐to‐end joining; and (iii) formation of long palindromic linear plasmids that duplicate the intact telomere by a non‐recombinational process. The relative frequency of use of these survival mechanisms depended on the location and length of the telomeric DNA deletion. Repair by intermolecular recombination between the telomeres of chromosomes and plasmids, deletion of additional DNA during plasmid circularization, and insertion of chromosomal DNA fragments into plasmids during end‐to‐end joining were observed. Our results show that damage to telomeres of Streptomyces linear replicons can promote major structural transformations in these replicons as well as genetic exchange between chromosomes and extrachromosomal DNA. Our findings also suggest that spontaneous circularization of linear Streptomyces chromosomes may be a biological response to instances of telomere damage that cannot be repaired by homologous recombination.
The Neurospora crassa pheromone precursor genes are regulated by the mating type locus and the circadian clock
Authors: Bobrowicz Piotr , Pawlak Rebecca , Correa Alejandro , Bell‐Pedersen Deborah , Ebbole Daniel J. 
Published: March 2003

SummaryPheromones play important roles in female and male behaviour in the filamentous ascomycete fungi. To begin to explore the role of pheromones in mating, we have identified the genes encoding the sex pheromones of the heterothallic species Neurospora crassa. One gene, expressed exclusively in mat A strains, encodes a polypeptide containing multiple repeats of a putative pheromone sequence bordered by Kex2 processing sites. Strains of the opposite mating type, mat a, express a pheromone precursor gene whose polypeptide contains a C‐terminal CAAX motif predicted to produce a mature pheromone with a C‐terminal carboxy‐methyl isoprenylated cysteine. The predicted sequences of the pheromones are remarkably similar to those encoded by other filamentous ascomycetes. The expression of the pheromone precursor genes is mating type specific and is under the control of the mating type locus. Furthermore, the genes are highly expressed in conidia and under conditions that favour sexual development. Both pheromone precursor genes are also regulated by the endogenous circadian clock in a time‐of‐day‐specific fashion, supporting a role for the clock in mating.
Mutations in Bacillus subtilis glutamine synthetase that block its interaction with transcription factor TnrA
Authors: Fisher Susan H. , Brandenburg Jaclyn L. , Wray Lewis V. 
Published: March 2003

SummaryIn Bacillus subtilis, the activity of the nitrogen regulatory factor TnrA is regulated through a protein– protein interaction with glutamine synthetase. During growth with excess nitrogen, the feedback‐inhibited form of glutamine synthetase binds to TnrA and blocks DNA binding by TnrA. Missense mutations in glutamine synthetase that constitutively express the TnrA‐regulated amtB gene were characterized. Four mutant proteins were purified and shown to be defective in their ability to inhibit the in vitro DNA‐binding activity of TnrA. Two of the mutant proteins exhibited enzymatic properties similar to those of wild‐type glutamine synthetase. A model of B. subtilis glutamine synthetase was derived from a crystal structure of the Salmonella typhimurium enzyme. Using this model, all the mutated amino acid residues were found to be located close to the glutamate entrance of the active site. These results are consistent with the glutamine synthetase protein playing a direct role in regulating TnrA activity.
Donor strand complementation mechanism in the biogenesis of non‐pilus systems
Authors: Zavialov A. V. , Kersley J. , Korpela T. , Zav’yalov V. P. , MacIntyre S. , Knight S. D. 
Published: March 2003

SummaryThe F1 antigen of Yersinia pestis belongs to a class of non‐pilus adhesins assembled via a classical chaperone–usher pathway. Such pathways consist of PapD‐like chaperones that bind subunits and pilot them to the outer membrane usher, where they are assembled into surface structures. In a recombinant Escherichia coli model system, chaperone–subunit (Caf1M:Caf1n) complexes accumulate in the periplasm. Three inde‐pendent methods showed that these complexes are rod‐ or coil‐shaped linear arrays of Caf1 subunits capped at one end by a single copy of Caf1M chaperone. Deletion and point mutagenesis identified an N‐terminal donor strand region of Caf1 that was essential for polymerization in vitro, in the periplasm and at the cell surface, but not for chaperone–subunit interaction. Partial protease digestion of periplasmic complexes revealed that this region becomes buried upon formation of Caf1:Caf1 contacts. These results show that, despite the capsule‐like appearance of F1 antigen, the basic structure is assembled as a linear array of subunits held together by intersubunit donor strand complementation. This example shows that strikingly different architectures can be achieved by the same general principle of donor strand complementation and suggests that a similar basic polymer organization will be shared by all surface structures assembled by classical chaperone–usher pathways.
Stage‐specific gene expression during sexual development in Phytophthora infestans
Authors: Fabritius Anna‐Liisa , Cvitanich Cristina , Judelson Howard S. 
Published: March 2003

SummaryEight genes that are upregulated during sexual development in the heterothallic oomycete, Phytophthora infestans, were identified by suppression subtractive hybridization. Two genes showed very low but detectable expression in vegetative hyphae and became induced about 40‐ to >100‐fold early in mating, before gametangial initials appeared. The remaining six loci were not induced until later in mating, coincident with the formation of gametangia and oospores, with induction levels ranging from 60‐ to >100‐fold. Five genes were single copy, and three were members of families. Sequence analysis revealed that the predicted products of three of the genes had similarity to proteins that influence RNA stability, namely a ribonuclease activator, the pumilio family of RNA‐binding proteins and RNase H. The products of two other mating‐induced genes resembled two types of Phytophthora proteins previously shown to elicit plant defence responses. Each mating‐induced gene was also expressed in a self‐fertile strain, which was shown to be a heterokaryon. However, quantitative and qualitative differences existed in their expression in normal matings and in the self‐fertile heterokaryon. Besides the mating‐induced genes, two extrachromosomal RNA elements were identified.
The GlnD and GlnK homologues of Streptomyces coelicolor A3(2) are functionally dissimilar to their nitrogen regulatory system counterparts from enteric bacteria
Authors: Hesketh A. , Fink D. , Gust B. , Rexer H.‐U. , Scheel B. , Chater K. , Wohlleben W. , Engels A. 
Published: March 2003

SummaryGlutamine synthetase I (GSI) enzyme activity in Streptomyces coelicolor is controlled post‐translationally by the adenylyltransferase (GlnE) as in enteric bacteria. Although other homologues of the Escherichia coli Ntr system (glnK, coding for a PII family protein; and glnD, coding for an uridylyltransferase) are found in the S. coelicolor genome, the regulation of the GSI activity was found to be different. The functions of glnK and glnD were analysed by specific mutants. Surprisingly, biochemical assay and two‐dimensional PAGE analysis showed that modification of GSI by GlnE occurs normally in all mutant strains, and neither GlnK nor GlnD are required for the regulation of GlnE in response to nitrogen stimuli. Analysis of the post‐translational regulation of GlnK in vivo by two‐dimensional PAGE and mass spectrometry indicated that it is subject to both a reversible and a non‐reversible modification in a direct response to nitrogen availability. The irreversible modification was identified as removal of the first three N‐terminal amino acid residues of the protein, and the reversible modification as adenylylation of the conserved tyro‐sine 51 residue that is known to be uridylylated in E. coli. The glnD insertion mutant expressing only the N‐terminal half of GlnD was capable of adenylylating GlnK, but was unable to perform the reverse deadenylylation reaction in response to excess ammonium. The glnD null mutant completely lacked the ability to adenylylate GlnK. This work provides the first example of a PII protein that is modified by adenylylation, and demonstrates that this reaction is performed by a homologue of GlnD, previously described only as a uridylyltransferase enzyme.
Molecular characterization and analysis of the biosynthetic gene cluster for the azoxy antibiotic valanimycin
Authors: Garg Ram P. , Ma Yunqing , Hoyt Jeffrey C. , Parry Ronald J. 
Published: March 2003

SummaryStreptomyces viridifaciens
MG456‐hF10 produces the antibiotic valanimycin, a naturally occurring azoxy compound. Valanimycin is known to be derived from valine and serine with the intermediacy of isobutylamine and isobutylhydroxylamine, but little is known about the stages in the pathway leading to the formation of the azoxy group. In previous studies, a cosmid containing
S. viridifaciens
DNA was isolated that conferred valanimycin production upon
Strepto‐myces lividans
TK24. Subcloning of DNA from the valanimycin‐producing cosmid has led to the identi‐fication of a 22 kb segment of DNA sufficient to allow valanimycin production in
S. lividans
TK24. Sequencing of this DNA segment and the surrounding DNA revealed the presence of 20 genes. Gene disruption experiments defined the boundaries of the valanimycin gene cluster, which appears to contain 14 genes. The cluster includes an amino acid decar‐boxylase gene (
vlmD
), a valanimycin resistance gene (
vlmF
), at least two regulatory genes (
vlmE
,
vlmI
), two genes encoding a flavin monooxygenase (
vlmH
,
vlmR
), a seryl tRNA synthetase gene (
vlmL
) and seven genes of unknown function. Overproduction and characterization of VlmD demonstrated that it catalyses the decarboxylation of
l ‐valine. An unusual feature of the valanimycin gene cluster is that four genes involved in branched amino acid biosynthesis are located near its 5′ end.
Characterization of interaction sites in the Saccharomyces cerevisiae ribosomal stalk components
Authors: Lalioti V. S. , Pérez‐Fernández J. , Remacha M. , Ballesta J. P. G. 
Published: March 2003

SummaryThe interactions among the yeast stalk components (P0, P1α, P1β, P2α and P2β) and with EF‐2 have been explored using immunoprecipitation, affinity chromatography and the two‐hybrid system. No stable association was detected between acidic proteins of the same type. In contrast, P1α and P1β were found to interact with P2β and P2α respectively. An interaction of P0 with P1 proteins, but not with P2 proteins, was also detected. This interaction is strongly increased with the P0 carboxyl end, which is able to form a pentameric complex with the four acidic proteins. The P1/P2 binding site has been located between residues 212 and 262 using different C‐terminal P0 fragments. Immunoprecipitation shows the association of EF‐2 with protein P0. However, the interaction is stronger with the P1/P2 proteins than with P0 in the two‐hybrid assay. This interaction improves using the 100‐amino‐acid‐long C‐end of P0 and is even higher with the last 50 amino acids. The data indicate a specific association of P1α with P2β and of P1β with P2α rather than the dimerization of the acidic proteins found in prokaryotes. In addition, they suggest that stalk assembly begins by the interaction of the P1 proteins with P0. Moreover, as functional interactions of the complete P0 were found to increase using protein fragments, the data suggest that some active sites are exposed in the ribosome as a result of conformational changes that take place during stalk assembly and function.
The UV‐B stimulon of the terrestrial cyanobacterium Nostoc commune comprises early shock proteins and late acclimation proteins
Authors: Ehling‐Schulz Monika , Schulz Stefan , Wait Robin , Görg Angelika , Scherer Siegfried 
Published: March 2003

SummaryThe UV‐B and desiccation‐tolerant terrestrial cyanobacterium Nostoc commune was grown under defined UV irradiation. Proteome changes were monitored in the membrane and the cytosolic and the extracellular fractions. Tools were developed to separate stress‐triggered from growth stage‐dependent changes. UV‐B changed the relative cellular concentration of 493 out of 1350 protein spots at least by a factor of three, rendering the UV‐B stimulon of N. commune the most complex one described so far. It comprises two different parts: an early shock response influencing 214 proteins and a late accli‐mation response involving 279 proteins. The shock response comprised many membrane or membrane‐associated proteins, whereas the acclimation re‐sponse mainly changed cytosolic proteins. Most of the shock‐induced changes were transient and did not overlap with the acclimation response. In the extracellular fraction, UV irradiation induced superoxide dismutase and the water stress protein. In total, 27 intracellular, UV‐B‐induced proteins were partially sequenced by electrospray ionization tandem mass spectrometry. Three functional classes were identified: proteins involved in lipid metabolism, in carbohydrate metabolism and in regulatory pathways. About 50% of the sequenced proteins were homologous to cyanobacterial database entries with un‐known function. Interestingly, all of these proteins belong to the UV‐B acclimation response. We conclude that the UV‐B shock response and the UV‐B acclimation response represent two completely different and remarkably complex strategies of N. commune to protect itself against UV‐B radiation in its natural environment.
The histone deacetylase Hda1 from Ustilago maydis is essential for teliospore development
Authors: Reichmann Michael , Jamnischek Alexander , Weinzierl Gerhard , Ladendorf Oliver , Huber Sieglinde , Kahmann Regine , Kämper Jörg 
Published: March 2003

SummaryIn the corn smut fungus Ustilago maydis, pathogenic development is controlled by the b mating type locus that encodes the two homeodomain proteins bE and bW. A heterodimer of bE and bW controls a large set of genes, either directly by binding to cis regulatory sequences or indirectly via a b‐dependent regulatory cascade. It is thought that several of the b‐regulated genes contribute to processes involved in pathogenicity. In a screen for components of the b‐dependent regulatory cascade we have isolated Hda1, a protein with homology to histone deacetylases of the RPD3 class. Hda1 can substitute for the histone deacetylase RPD3 in Saccharomyces cerevisiae, showing that it functions as a histone deacetylase. Deletion of hda1 results in the expression of several genes that are normally expressed only in the dikaryon, among these are several genes that are now expressed independently from their activation by the bE/bW heterodimer. hda1 mutant strains are capable to infect corn, and the proliferation of dikaryotic hyphae within the plant appears comparable to wild‐type strains during initial developmental stages. Upon karyogamy, however, the proliferation to mature teliospores is blocked. The block in sporogenesis in Δhda1 strains is probably a result of the deregulation of a specific set of genes whose temporal or spatial expression prevent the proper developmental progress.
Motility modes of Spiroplasma melliferum BC3: a helical, wall‐less bacterium driven by a linear motor
Authors: Gilad Rami , Porat Asher , Trachtenberg Shlomo 
Published: March 2003

Summary Spiroplasma
are members of the Mollicutes (
Mycoplasma, Acholeplasma
and
Spiroplasma
) – the simplest, minimal, free‐living and self‐replicating forms of life. The mollicutes are unique among bacteria in completely lacking cell walls and flagella and in having an internal, contractile cytoskeleton, which also functions as a linear motor.
Spiroplasma are
helical, chemotactic and viscotactic active swimmers. The Spiroplasmal cytoskeleton is a flat ribbon composed of seven pairs of fibrils. The ribbon is attached to the inner side of the cell membrane along its innermost (shortest) helical line. The cell's geometry and dynamic helical parameters, and consequently motility, can be controlled by changing differentially and in a co‐ordinated manner, the length of the fibrils. We identified several consistent modes of cell movements and motility originating, most likely, as a result of co‐operative or local molecular switching of fibrils: (i) regular extension and contraction within the limits of helical symmetry (this mode also includes straightening, beyond what is allowed by helical symmetry, and reversible change of helical sense); (ii) spontaneous and random change of helical sense originating at random sites along the cell (these changes propagate along the cell in either direction and hand switching is completed within ∼0.08 second); (iii) forming a deformation on one of the helical turns and propagating it along the cell (these helical deformations may travel along the cell at a speed of up to ∼40 µm s
−1
); (iv) random bending, flexing and twitching (equivalent to tumbling). In standard medium (viscosity = 1.147 centipoise) the cells run at ∼1.5 µm s
−1
, have a Reynolds number of ∼3.5 × 10
−6
and consume ∼30 ATP molecules s
−1 . Running velocity, duration, persistence and efficiency increase with viscosity upon adding ficoll, dextran and methylcellulose to standard media. Relative force measurements using optical tweezers confirm these findings.