bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inactivation of the decay pathway initiated at an internal site by RNase E promotes poly(A)‐dependent degradation of the rpsO mRNA in Escherichia coli
Authors: Marujo Paulo E. , Braun Frédérique , Haugel‐Nielsen Jeanette , Le Derout Jacques , Arraiano Cecília M. , Régnier Philippe 
Published: November 2003

SummaryIn Escherichia coli, RNA degradation is mediated by endonucleolytic processes, frequently mediated by RNase E, and also by a poly(A)‐dependent mechanism. The dominant pathway of decay of the rpsO transcripts is initiated by an RNase E cleavage occurring at a preferential site named M2. We demonstrate that mutations which prevent this cleavage slow down degradation by RNase E. All these mutations reduce the single‐stranded character of nucleotides surrounding the cleavage site. Moreover, we identify two other cleavage sites which probably account for the slow RNase E‐mediated degradation of the mutated mRNAs. Failure to stabilize the rpsO transcript by appending a 5′ hairpin indicates that RNase E is not recruited by the 5′ end of mRNA. The fact that nucleotide substitutions which prevent cleavage at M2 facilitate the poly(A)‐dependent degradation of the rpsO transcripts suggest an interplay between the two mechanisms of decay. In the discussion, we speculate  that  a  structural  feature  located  in  the  vicinity of M2 could be an internal degradosome entry site promoting both RNase E cleavages and poly(A)‐dependent degradation of the rpsO mRNA. We also discuss the role of poly(A)‐dependent decay in mRNA metabolism.
Fitness effects of DHFR‐TS mutations associated with pyrimethamine resistance in apicomplexan parasites
Authors: Fohl Leah M. , Roos David S. 
Published: November 2003

SummaryPyrimethamine resistance in the malaria parasite Plasmodium falciparum is characterized by specific point mutations in the dihydrofolate reductase (DHFR) domain of the bifunctional dihydrofolate reductase‐thymidylate synthase (DHFR‐TS) gene. We have previously explored the effect of these mutations by engineering homologous alleles of Toxoplasma gondii DHFR‐TS, which can readily be expressed as recombinant protein for enzymatic studies, or as allelic replacements in transgenic parasites. In order to directly assess the costs of pyrimethamine‐resistance in vivo, we have carried out competition studies between mixtures of T. gondii tachyzoites harbouring wild‐type or mutant DHFR‐TS alleles, both in tissue culture and in mice. Arg59+Asn108 mutants (using the P. falciparum numbering system) exhibit no significant fitness defects in vitro, but a fitness defect of 1.8% per generation in mice. Arg59+Ser223 mutants exhibit fitness defects of > 2.8% per generation both in vitro and in vivo, which may explain why this highly pyrimethamine‐resistant allele has not been observed in the field. It is important to note that long‐term propagation of parasites in vitro or in vivo can produce adaptations affecting fitness by > 3.7% per generation, necessitating careful attention to background in head‐to‐head competition studies. A sensitive PCR‐based assay permits different growth rates to be assessed even in the absence of a drug resistance marker that can be scored by plaque assay.
Cleavage of preflagellins by an aspartic acid signal peptidase is essential for flagellation in the archaeon Methanococcus voltae
Authors: Bardy Sonia L. , Jarrell Ken F. 
Published: November 2003

SummaryThe differences between archaeal and bacterial flagella are becoming more apparent as research on the archaeal structure progresses. One crucial difference is the presence of a leader peptide on archaeal preflagellins, which is removed from the flagellin prior to its incorporation into the flagellar filament. The enzyme responsible for the removal of the flagellin leader peptide was identified as FlaK. FlaK of Methanococcus voltae retains its preflagellin peptidase activity when expressed in Escherichia coli and used in an in vitro assay. Homologous recombination of an integration vector into the chromosomal copy of flaK resulted in a non‐motile, non‐flagellated phenotype. The flagellins of the mutant had larger molecular weights than their wild‐type counterparts, as expected if they retained their 11‐ to 12‐amino‐acid leader peptide. Membranes of the flaK mutant were unable to process preflagellin in the in vitro assay. Site‐directed mutagenesis demonstrated that two aspartic acid residues conserved with ones in type IV prepilin peptidases were necessary for proper recognition or processing of the preflagellin. As bacterial flagellins lack a leader peptide and a peptidase is not required for export and assembly, the requirement for FlaK further emphasizes the similarity archaeal flagella have with type IV pili, rather than with bacterial flagella.
amfR, an essential gene for aerial mycelium formation, is a member of the AdpA regulon in the A‐factor regulatory cascade in Streptomyces griseus
Authors: Yamazaki Haruka , Takano Yuji , Ohnishi Yasuo , Horinouchi Sueharu 
Published: November 2003

SummaryIn Streptomyces griseus, A‐factor (2‐isocapryloyl‐3R‐hydroxymethyl‐γ‐butyrolactone) acts as a chemical signalling molecule that triggers morphological differentiation and secondary metabolism. A transcriptional activator, AdpA, in the A‐factor regulatory cascade switches on a number of genes required for both processes, thus forming an AdpA regulon. amfR encoding a regulatory protein similar to response regulators of bacterial two‐component regulatory systems and essential for aerial mycelium formation was found to be a member of the AdpA regulon. AdpA bound two sites at nucleotide positions approximately −200 (site 1) and −60 (site 2), with respect to the major transcriptional start point of amfR, and accelerated the transcription of amfR by assisting RNA polymerase in forming an open complex at an appropriate region including the transcriptional start point. Site 2 contributed more to the transcriptional activation of amfR by AdpA than site 1, although AdpA showed a much lower affinity to site 2 than to site 1. The amfR transcription enhanced by AdpA subsequently ceased at day 2 when aerial hyphae began to be formed in the wild‐type strain, whereas in an adsA null mutant amfR was continuously transcribed even until day 3. This implied that amfR was repressed growth dependently by a gene product under the control of σ‐AdsA. Transcription of the promoter upstream of amfT depended on amfR, which is consistent with the idea that AmfR serves as an activator for amfTSBA in the amf operon. The observations that the amfR gene contains a TTA codon, a potential target for bldA‐mediated regulation, and a conserved Asp‐54 residue, which might be phosphorylated by a sensor kinase, suggest that the amf operon is under transcriptional, translational and post‐translational control systems.
Cold shock induction of RNase R and its role in the maturation of the quality control mediator SsrA/tmRNA
Authors: Cairrão Fátima , Cruz Ana , Mori Hiroyuki , Arraiano Cecília Maria 
Published: November 2003

SummaryIn this paper we show that RNase R is a cold shock protein that is induced seven‐ to eightfold by cold shock and that its expression is tightly regulated by temperature. Transcriptional studies reveal that the rnr gene is co‐transcribed with flanking genes as an operon induced under cold shock. The induction of RNase R levels is mainly a result of the stabilization of the rnr transcripts. The transient stability of the rnr transcripts is shown to be regulated by PNPase at the end of the acclimation phase. Studies with an rnr mutant revealed a cold‐shock phenotype showing that RNase R contributes to growth at low temperatures. We have shown that RNase R can be involved in the maturation of SsrA/tmRNA, an important small stable RNA involved in protein tagging and ribosome rescue. The wide biological significance of RNase R regarding adaptation to cold shock and its involvement in RNA surveillance, protein quality control and pathogenesis is discussed.
Yersinia yopQ mRNA encodes a bipartite type III secretion signal in the first 15 codons
Authors: Ramamurthi Kumaran S. , Schneewind Olaf 
Published: November 2003

SummaryThe type III machinery of Yersinia transports Yop proteins across the bacterial envelope. The minimal secretion signal of yopQ is located in codons 1–10 that, when fused in frame to the neomycin phosphotransferase gene, is sufficient to promote type III secretion of YopQ1–10‐Npt. Frame‐shift mutations, generated by nucleotide insertions or deletions following the AUG start and suppressed at the fusion site with npt, abrogate signalling of yopQ1–10 but not of yopQ1–15. By generating transversions of every single nucleotide in yopQ1–10, we identified 10 nucleotide positions in codons 2, 3, 5, 7, 9 and 10 that were each required for substrate recognition. One transversion that abolishes secretion, uridyl 9 to adenyl (U9A), is a synonymous codon 3 mutation that retains the original amino acid as confirmed by Edman degradation analysis, suggesting that the mRNA but not the amino acid sequence of yopQ1–10 is involved in secretion signalling. Although transversion of U8A abrogates signalling of yopQ1–10, fusion of yopQ codons 11–15 restores secretion. The nucleotides that are required for this suppression by yopQ11–15 were identified and revealed both synonymous and non‐synonymous mutations. Frame‐shift mutations introduced into just this suppressor region (codons 11–15) did not abrogate its ability to suppress mutations in the minimal secretion signal (codons 1–10). Thus, elements downstream of the minimal secretion signal of YopQ increase the efficiency of YopQ secretion and suppress mutations elsewhere in the secretion signal.
The quaternary structure of RNase G from Escherichia coli
Authors: Briant Douglas J. , Hankins Janet S. , Cook Michael A. , Mackie George A. 
Published: November 2003

SummaryRNase G is the endoribonuclease responsible for forming the mature 5′ end of 16S rRNA. This enzyme shares 35% identity with and 50% similarity to the N‐terminal 470 amino acids encompassing the catalytic domain of RNase E, the major endonuclease in Escherichia coli. In this study, we developed non‐denaturing purifications for overexpressed RNase G. Using mass spectrometry and N‐terminal sequencing, we unambiguously identified the N‐terminal sequence of the protein and found that translation is initiated at the second of two potential start sites. Using velocity sedimentation and oxidative cross‐linking, we determined that RNase G exists largely as a dimer in equilibrium with monomers and higher multimers. Moreover, dimerization is required for activity. Four of the six cysteine residues of RNase G were mutated to serine. No single cysteine to serine mutation resulted in a complete loss of cross‐linking, dimerization or activity. However, multiple mutations in a highly conserved cluster of cysteines, including C405 and C408, resulted in a partial loss of activity and a shift in the distribution of RNase G multimers towards monomers. We propose that many of the cysteines in RNase G lie on its surface and define, in part, the subunit–subunit interface.
Substrate recognition by the Yersinia type III protein secretion machinery
Authors: Ramamurthi Kumaran S. , Schneewind Olaf 
Published: November 2003

SummaryType III secretion is the designation given to those protein secretion pathways, primarily in pathogenic Gram‐negative bacteria, whose secretion machinery components share an amino acid sequence homology to components of the flagellar basal body. In Yersinia spp., these secretion machineries inject virulence proteins called Yops into the cytosol of target macrophages in an effort to evade phagocytic killing. To date, a clear mechanism by which Yops are recognized by the type III secretion machinery has not been elucidated. Unlike most, if not all, previously characterized protein sorting pathways, the information that identifies Yops as substrates for secretion seems not to be wholly encoded within the Yop peptide sequence. In fact, it appears that at least some of this information is contained within yop mRNAs. This review summarizes recent observations that have been made in this unusual field and proposes models by which proteins may be initiated into this pathway.
Characterization of the hipA7 allele of Escherichia coli and evidence that high persistence is governed by (p)ppGpp synthesis
Authors: Korch Shaleen B. , Henderson Thomas A. , Hill Thomas M. 
Published: November 2003

SummaryThe ability of a high frequency (10−2) of Escherichia coli to survive prolonged exposure to penicillin antibiotics, called high persistence, is associated with mutations in the hipA gene. The hip operon is located in the chromosomal terminus near dif and consists of two genes, hipA and hipB. The wild‐type hipA gene encodes a toxin, whereas hipB encodes a DNA‐binding protein that autoregulates expression of the hip operon and binds to HipA to nullify its toxic effects. We have characterized the hipA7 allele, which confers high persistence, and established that HipA7 is non‐toxic, contains two mutations (G22S and D291A) and that both mutations are required for the full range of phenotypes associated with hip mutants. Furthermore, expression of hipA7 in the absence of hipB is sufficient to establish the high persistent phenotype, indicating that hipB is not required. There is a strong correlation between the frequency of persister cells generated by hipA7 strains and cell density, with hipA7 strains generating a 20‐fold higher frequency of persisters as cultures approach stationary phase. It is also demonstrated that relA knock‐outs diminish the high persistent phenotype in hipA7 mutants and that relA spoT knock‐outs eliminate high persistence altogether, suggesting that hipA7 facilitates the establishment of the persister state by inducing (p)ppGpp synthesis. Consistent with this proposal, ectopic expression of relA′ from a plasmid was shown to increase the number of persistent cells produced by hipA7 relA double mutants by 100‐fold or more. A model is presented that postulates that hipA7 increases the basal level of (p)ppGpp synthesis, allowing a significantly greater percentage of cells in a population to assume a persistent, antibiotic‐insensitive state by potentiating a rapid transition to a dormant state upon application of stress.
Cell wall α‐1,3‐glucan is required to anchor the Cryptococcus neoformans capsule
Authors: Reese Amy J. , Doering Tamara L. 
Published: November 2003

Summary Cryptococcus neoformans is an opportunistic pathogen responsible for serious disease in humans. Critical for virulence of this fungus is an elaborate polysaccharide capsule, which impedes the host immune response. We found that association of the capsule with the cell requires a specific component of the cell wall, α‐1,3‐glucan. Post‐transcriptional inhibition of α‐1,3‐glucan synthase expression, using double‐stranded RNA interference, yields cells that are unable to assemble a capsule although they generate its polysaccharide components. The resulting cryptococci are slow‐growing and acapsular. This finding demonstrates a novel mode of polysaccharide attachment and an important application of RNA interference in fungi. The elimination of the capsule by reducing the expression of a single gene suggests a potential avenue for antifungal chemotherapy.
Assembly of pili on the surface of Corynebacterium diphtheriae
Authors: Ton‐That Hung , Schneewind Olaf 
Published: November 2003

SummaryPili of Gram‐negative pathogens are formed from pilin precursor molecules by non‐covalent association within the outer membrane envelope. Gram‐positive microbes employ the cell wall peptidoglycan as a surface organelle for the covalent attachment of proteins, however, an assembly pathway for pili has not yet been revealed. We show here that pili of Corynebacterium diphtheriae are composed of three pilin subunits, SpaA, SpaB and SpaC. SpaA, the major pilin protein, is distributed uniformly along the pilus shaft, whereas SpaB is observed at regular intervals and SpaC seems positioned at the pilus tip. Assembled pili are released from the bacterial surface by treatment with murein hydrolase, suggesting that the pilus fibres may be anchored to the cell wall envelope. All three pilin subunit proteins are synthesized as precursors carrying N‐terminal signal peptides and C‐terminal sorting signals. Some, but not all, of the six sortase genes encoded in the genome of C. diphtheriae are required for precursor processing, pilus assembly or cell wall envelope attachment. Pilus assembly is proposed to occur by a mechanism of ordered cross‐linking, whereby pilin‐specific sortase enzymes cleave precursor proteins at sorting signals and involve the side chain amino groups of pilin motif sequences to generate links between pilin subunits. This covalent tethering of adjacent pilin subunits appears to have evolved in many Gram‐positive pathogens that encode sortase and pilin subunit genes with sorting signals and pilin motifs.
Drug‐induced alterations in gene expression of the asexual blood forms of Plasmodium falciparum
Authors: Gunasekera Anusha Munasinghe , Patankar Swati , Schug Jonathan , Eisen Geoffrey , Wirth Dyann F. 
Published: November 2003

SummaryThe complete genome sequence of the malarial parasite, Plasmodium falciparum[Gardner, M.J., Hall, N., Fung, E., White, O., Berriman, M., and Hyman, R.W. (2002) Nature 419: 498–510; Hyman, R.W., Fung, E., Conway, A., Kurdi, O., Mao, J., Miranda, M. et al. (2002) Nature 419: 534–537], has provided researchers with the informational base for establishing genomic [Volkman, S.K., Hartl, D.L., Wirth, D.F., Nielsen, K.M., Choi, M., Batalov, S., et al. (2002) Science 298: 216–218], proteomic [Florens, L., Washburn, M.P.J.D.R., Anthony, R.M., Grainger, M., Haynes, J.D., et al. (2002) Nature 419: 520–526; Lasonder, E., Ishihama, Y., Anderson, J.S., Vermunt, A.M.W., Pain, A., Sauerwein, R.W., et al. (2002) Nature 419: 537–542] and genome‐wide RNA expression [Ben Mamoun, C., Gluzman, I.Y., Hott, C., MacMillan, S.K., Amarakone, A.S., Anderson, D.L., et al. (2001) Mol Microbiol 39: 26–36; Hayward, R.E., Derisi, J.L., Alfadhli, S., Kaslow, D.C., Brown, P.O., and Rathod, P.K. (2000) Mol Microbiol 35: 6–14] analyses in this system. In fact, we have previously utilized SAGE (serial analysis of gene expression) to identify abundant loci that probably constitute part of the active metabolome [Patankar, S., Munasinghe, A., Shoaibi, A., Cummings, L.M., and Wirth, D.F. (2001) Mol Biol Cell 12: 3114–3125], as well as to characterize antisense transcription on a global scale in Plasmodium. In the present study, the comprehensive annotation of SAGE libraries derived from an asexual stage population exposed to drug and its matched control was used to assess the modulation of gene expression by chloroquine. Here, we observed a constellation of changes, with the differential regulation of over 100 transcripts, and have confirmed the data by alternate methods. A few responsive loci, including PfMDR1, have previously been implicated in the mechanism of chloroquine action/resistance. Several others, however, were derived from unexpected categories, including a large number of unknown open reading frames (ORFs), whose induction after drug exposure may provide first hints to their possible function.
Discovery of two northern hemisphere Armillaria species on Proteaceae in South Africa
Authors: Coetzee M. P. A. , Wingfield B. D. , Roux J. , Crous P. W. , Denman S. , Wingfield M. J. 
Published: November 2003

Armillaria root rot symptoms were observed on native Protea and Leucadendron (Proteaceae) species in Kirstenbosch Botanical Gardens in the Western Cape Province of South Africa. Intergenic spacer (IGS)‐1 polymerase chain reaction (PCR) restriction fragment‐length polymorphism (RFLP) profiling indicated the presence of at least two Armillaria species. The profiles of two isolates were identical to those of A. mellea s. str., originating in Europe. Phylogenetic analyses incorporating internal transcribed spacer (ITS) and IGS‐1 sequence data identified the remaining isolates as closely related to A. calvescens, A. gallica, A. jezoensis and A. sinapina. These isolates displayed mating compatibility with A. gallica. From the RFLP profiles, sequencing results and sexual compatibility studies, it is concluded that the two species on Proteaceae in Kirstenbosch represent A. mellea and A. gallica. These are northern hemisphere fungi that have apparently been accidentally introduced into South Africa. This is the second report of Armillaria being introduced into South Africa. The introduction probably occurred early in the colonization of Cape Town, when potted plants from Europe were used to establish gardens.
Development of natural late blight epidemics in pure and mixed plots of potato cultivars with different levels of partial resistance
Authors: Andrivon D. , Lucas J.‐M. , Ellissèche D. 
Published: November 2003

Late blight, caused by Phytophthora infestans, is the most severe disease of potato worldwide. Controlling late blight epidemics is difficult, and resistance of host cultivars is either not effective enough, or too easily overcome by the pathogen to be used alone. In field trials conducted for 3 years under natural epidemics, late blight severity was significantly lower in a susceptible cultivar growing in rows alternating with partially resistant cultivars (mixtures) than in unmixed plots of the susceptible cultivar alone. Partially resistant cultivars behaved similarly in unmixed and mixed plots. Mixtures of cultivars reduced disease progress rates and sometimes delayed disease onset over unmixed plots, but did so significantly only for the slowest epidemic. This suggests that reduction of area under the disease progress curve (AUDPC) in mixtures resulted from the cumulative action of minor effects. Disease distribution was focal in all plots at all dates, as shown by Morisita's index values significantly exceeding 1. Significant yield increases for the susceptible cultivar, and occasionally for the partially resistant ones, were observed in mixed‐cultivar plots compared with single‐cultivar plots. These results show that cultivar mixtures can significantly reduce natural, polycyclic epidemics in broadleaved plants attacked by pathogens causing rapidly expanding lesions.