bluuebunny/crossref_metadata_2025 huggingface dataset:
DARTS 2000 online diabetes management system: formative evaluation in clinical practice
Authors: Pagliari Claudia , Clark Deborah , Hunter Karen , Boyle Douglas , Cunningham Scott , Morris Andrew , Sullivan Frank
Published: November 2003

Rationale, aims and objectives  Failure to engage in user‐informed evaluation of emergent health informatics tools can have negative consequences for future implementation, related both to poor usability or clinical utility and to suboptimal stakeholder buy‐in. This paper describes a formative evaluation in primary care of a multifaceted, web‐based resource for diabetes management. The primary aims were to assess the usability and utility of the prototype in order to inform system refinements prior to implementation, and  to  investigate  barriers  and facilitators to system use so as to aid the development of a tailored implementation plan.

Methods  A mixed‐method approach involving survey, remote observation, semi‐structured interviews and electronic feedback.

Context  One Scottish local health care cooperative comprising five general practice surgeries and their staff.

Results  A survey following temporary exposure to a dummy site revealed high levels of computer familiarity, welcoming attitudes and positive ratings of usability, format and utility. Comments mainly addressed content accuracy, feature suggestions and usability issues. Key barriers and facilitators to use included time and training. Remote observation following access to live clinical data enabled profiling of usage by individual and professional group. In the 3 month observation period administrators were the most frequent users, followed by GPs and nurses, with clinical and data entry screens accessed most often. Semi‐structured interviews with key respondents sampled by professional group and usage frequency provided richer qualitative information on barriers and facilitators to use, patterns of system integration into work routines and system usability, content and utility. Content analysis of electronic feedback revealed mainly technical queries and general expressions of satisfaction.

Conclusions  Evaluation informed a number of important and unforeseen improvements to the prototype and helped refine the implementation plan. Engagement in the process of evaluation has led to high levels of stakeholder ownership and widespread implementation.
Physician gender and antihypertensive prescription pattern in primary care
Authors: Sequeira Reginald P. , Al Khaja Khalid A. Jassim , Damanhori Awatif H. H. , Mathur Vijay S.
Published: November 2003

Objectives  To determine: (i) the gender‐based differences of physicians in prescribing antihypertensive drugs in the management of hypertension; (ii) the influence of patient comorbidity such as diabetes mellitus on the gender‐based pattern of antihypertensive prescription, and (iii) gender‐based prescription patterns among family physicians (FPs) and general practitioners (GPs).

Methods  A survey study was carried out at 15 out of 20 health centres in Bahrain during May and June 2000. A total of 3971 prescriptions, issued to 2705 patients with uncomplicated hypertension and 1266 patients with diabetic hypertension by 77 FPs (female = 54, male = 23) and 41 GPs (female = 11, male = 30), were analysed.

Results  As monotherapy, female physicians preferred ACE inhibitors (OR: 0.82, CI: 0.68–0.98, P = 0.033). In terms of overall drug utilization (monotherapy + combination therapy): (i) male physicians preferred beta blockers (OR: 1.17, CI: 1.03–1.31, P = 0.014) and diuretics (OR: 1.15, CI: 1.00–1.32, P = 0.047), while female physicians preferred methyldopa (OR: 0.73, CI: 0.56–0.94, P = 0.019); (ii) in uncomplicated hypertension, female physicians preferred calcium channel blockers (OR: 0.83, CI: 0.69–0.99, P = 0.038) and methyldopa (OR: 0.69, CI: 0.49–0.98, P = 0.042), and (iii) in diabetic hypertension, male physicians preferred beta blockers (OR: 1.26, CI: 1.00–1.57, P = 0.047). While female FPs prescribed methyldopa more extensively (OR: 0.66, CI: 0.47–0.92, P = 0.018), male GPs prescribed beta blockers (OR: 1.28, CI: 1.00–1.62, P = 0.046).

Conclusions  Within the same practice setting, gender‐based differences in the prescription of antihypertensive drugs were seen. Such preference for a particular class of antihypertensives was also influenced by the presence of comorbidity in patients and, to a limited extent, by the training level of primary care physicians.
Methodology for evaluating physician order entry (POE) implementations
Authors: Geiger Glen , Derman Yaron D.
Published: November 2003

The body of physician order entry (POE) implementations literature uses statistical evaluation methods to demonstrate changes in specified variables after POE implementation. To understand and manage the holistic impact of POE on the health care institution, a methodology that utilizes feedback to guide the POE implementation towards the satisfaction of stakeholder objectives is presented. Stakeholders jointly define quantitative and qualitative metrics for their objectives, establish target value vectors for the metrics that represent acceptable implementation outcomes and specify evaluation milestones. These are used to compare pre‐ and post‐POE implementation clinical performance, enabling a socio‐technical feedback–improvement cycle. A case study is provided to illustrate how the methodology is being used at Sunnybrook and Women's College Health Science Centre in Toronto, Canada.
Flavobacterium psychrophilum infections in salmonid fish
Authors: Nematollahi A , Decostere A , Pasmans F , Haesebrouck F
Published: November 2003

Flavobacterium psychrophilum is the causative agent of bacterial cold water disease and rainbow trout fry syndrome, disease entities responsible for substantial economic losses in salmonid aquaculture. Problems associated with epizootics include high mortality rate, increased susceptibility to other diseases, high labour costs of treatment and the enormous expenditure on chemotherapy. Despite the increasing significance of the disease, the pathogenesis of F. psychrophilum infections has only been partially elucidated, hampering the development of preventive measures to efficiently combat this disease condition. This literature review discusses the agent and the disease it causes, with emphasis on the bacterium–host interactions.
The HtrA family of serine proteases
Authors: Pallen Mark J. , Wren Brendan W.
Published: November 2003

HtrA, also known as DegP and probably identical to the Do protease, is a heat shock‐induced serine protease that is active in the periplasm of Escherichia coli. Homologues of HtrA have been described in a wide range of bacteria and in eukaryotes. Its chief role is to degrade misfolded proteins in the periplasm. Substrate recognition probably involves the recently described PDZ domains in the C‐terminal half of HtrA and, we suspect, has much in common with the substrate recognition system of the tail‐specific protease, Prc (which also possesses a PDZ domain). The expression of htrA is regulated by a complex set of signal transduction pathways, which includes an alternative sigma factor, RpoE, an anti‐sigma factor, RseA, a two‐component regulatory system, CpxRA, and two phosphoprotein phosphatases, PrpA and PrpB. Mutations in the htrA genes of Salmonella, Brucella and Yersinia cause decreased survival in mice and/or macrophages, and htrA mutants can act as vaccines, as cloning hosts and as carriers of heterologous antigens.
Reduced polysialic acid capsule expression in Escherichia coli K1 mutants with chromosomal defects in kpsF
Authors: Cieslewicz Michael , Vimr Eric
Published: November 2003

Neuroinvasive Escherichia coli K1 synthesizes and assembles a polysialic acid capsule virulence factor on the external leaflet of the outer membrane. This capsule functions in pathogenesis by blocking non‐immune host defence mechanisms and acting as a relatively non‐immunogenic molecular mimic of the polysialic acid chains found in high concentrations on neural cell adhesion molecules of the human embryo and neonate. The synthetic, regulatory and export components for capsule expression are encoded in three functionally distinct gene blocks or regions of the 20 kb kps‘pathogenicity island’. These regions are organized as two convergently transcribed operons inserted into the monocistronic tRNA gene, pheV. The six genes of the so‐called region 1 operon are transcribed in the same direction as pheV, and at least four of these genes are required for polysialic acid export. Expression of this operon is thermoregulated by transcriptional control of its first gene, kpsF. To investigate the function of region 1 further, two independent chromosomal disruptions were engineered by inserting promoterless, terminatorless kanamycin or chloramphenicol resistance cassettes into the HindIII site of the kpsF coding sequence. The chromosomal insertions were regulated by temperature in the same way as the wild‐type operon, demonstrating that this control mechanism remained intact in these mutants. Chemical, immunological and ultrastructural microscopical methods demonstrated that full‐length polysialic acid chains were synthesized but not exported by the kpsF mutants. This phenotype was correlated with decreased plaque diameter when the mutants were infected with the capsule‐specific bacteriophage K1F. The export defect could not be complemented in trans with kpsF + containing its cis‐regulatory region because of titration of an apparent positive regulator of region 1 expression, whereas complementation was observed with a plasmid expressing kpsF from a physiologically irrelevant promoter. An N‐terminal polyhistidine peptide was attached to KpsF and used to purify the overproduced polypeptide. Antibodies raised against KpsF identified at least one of its paralogues in E. coli, GutQ, suggesting that KpsF and its homologues are membrane associated. The results indicate the requirement for a precise balance between region 1 components of the capsule export machinery, and that KpsF plays a positive role in the assembly, operation or regulation of this apparatus.
Identification of 3‐hydroxypalmitic acid methyl ester as a novel autoregulator controlling virulence in Ralstonia solanacearum
Authors: Flavier Albert B. , Clough Steven J. , Schell Mark A. , Denny Timothy P.
Published: November 2003

Expression of virulence genes in Ralstonia solanacearum, a phytopathogenic bacterium, is controlled by a complex regulatory network that integrates multiple signal inputs. Production of several virulence determinants is co‐ordinately reduced by inactivation of phcB, but is restored by growth in the presence of a volatile extracellular factor (VEF) produced by wild‐type strains of R. solanacearum. The VEF was purified from spent culture broth by distillation, solvent extraction, and liquid chromatography. Gas chromatography and mass spectroscopy identified 3‐hydroxypalmitic acid methyl ester (3‐OH PAME) as the major component in the single peak of VEF activity. Authentic 3‐OH PAME and the purified VEF were active at ≤1 nM, and had nearly equivalent specific activities for stimulating the expression of eps (the biosynthetic locus for extracellular polysaccharide) in a phcB mutant. Authentic 3‐OH PAME also increased the production of three virulence factors by a phcB mutant over 20‐fold to wild‐type levels, restored normal cell density‐associated expression of eps and increased expression of eps when delivered via the vapour phase. Reanalysis of the PhcB amino acid sequence suggested that it is a small‐molecule S‐adenosylmethionine‐dependent methyltransferase, which might catalyse synthesis of 3‐OH PAME from a naturally occurring fatty acid. Biologically active concentrations of extracellular 3‐OH PAME were detected before the onset of eps expression, suggesting that it is an intercellular signal that autoregulates virulence gene expression in wild‐type R. solanacearum. Other than acyl‐homoserine lactones, 3‐OH PAME is the only endogenous fatty acid derivative shown to be an autoregulator and may be the first example of a new family of compounds that can mediate long‐distance intercellular communication.
Role of architectural elements in combinatorial regulation of initiation of DNA replication in Escherichia coli
Authors: Polaczek Piotr , Kwan Kelvin , Liberles David A. , Campbell Judith L.
Published: November 2003

Bending of DNA is a prerequisite of site‐specific recombination and gene expression in many regulatory systems involving the assembly of specific nucleoprotein complexes. We have investigated how the uniquely clustered Dam methylase sites, GATCs, in the origin of Escherichia coli replication (oriC ) and their methylation status modulate the geometry of oriC and its interaction with architectural proteins, such as integration host factor (IHF), factor for inversion stimulation (Fis) and DnaA initiator protein. We note that 3 of the 11 GATC sites at oriC are strategically positioned within the IHF protected region. Methylation of the GATCs enhances IHF binding and alters the IHF‐induced bend at oriC. GATC motifs also contribute to intrinsic DNA curvature at oriC and the degree of bending is modulated by methylation. The IHF‐induced bend at oriC is further modified by Fis protein and IHF affinity for its binding site may be impaired by protein(s) binding to GATCs within the IHF site. Thus, GATC sites at oriC affect the DNA conformation and GATCs, in conjunction with the protein‐induced bends, are critical cis‐acting elements in specifying proper juxtapositioning of initiation factors in the early steps of DNA replication.
Analysis of random and site‐directed mutations in rhlI, a Pseudomonas aeruginosa gene encoding an acylhomoserine lactone synthase
Authors: Parsek Matthew R. , Schaefer Amy L. , Greenberg E. P.
Published: November 2003

The opportunistic human pathogen Pseudomonas aeruginosa possesses two cell density‐dependent genetic regulatory systems that control expression of a number of secreted virulence factors. These two systems, the lasI–lasR and rhlI–rhlR gene pairs, are members of the luxI–luxR family of quorum‐sensing signal generators and signal receptors. The rhlI gene in P. aeruginosa encodes a 201‐amino‐acid protein that catalyses the synthesis of an autoinducer, butyrylhomoserine lactone. Through a programme of random and site‐specific mutagenesis of rhlI we have gained a better understanding of how its protein product functions. Eight residues critical to butyrylhomoserine lactone synthesis by RhlI were identified by random mutagenesis, and all mapped to a conserved region that spans residues 24–104. Seven of the eight residues were charged amino acids and the other was a glycine. By using site‐specific mutagenesis we showed that an active‐site cysteine or serine was not required for butyrylhomoserine lactone synthesis, and that two conserved aromatic amino acids in the postulated active site region could be altered without complete loss of RhlI activity. Furthermore, two residues towards the C‐terminus that align with critical residues in LuxI can be altered in RhlI without loss of activity. These studies suggest that as opposed to the current models for acyl substrate binding to quorum‐sensing signal generators, charged amino acid residues participate directly in the catalysis of butyrylhomoserine lactone synthesis rather than cysteines, serines or hydrophobic amino acids.
Transcriptional analysis of the divergent cagAB genes encoded by the pathogenicity island of Helicobacter pylori
Authors: Spohn Gunther , Beier Dagmar , Rappuoli Rino , Scarlato Vincenzo
Published: November 2003

Helicobacter pylori strains isolated from most patients with peptic ulcer disease and adenocarcinoma express the vacuolating toxin VacA and contain a pathogenicity island named cag. The cag pathogenicity island codes for more than 40 putative proteins with features similar to bacterial secretion systems. One of these proteins, CagA, is an immunodominant antigen with unknown function encoded by the cagA gene. In the present study, we have analysed the functional promoter elements of the H. pylori cagA gene as well as of the divergently transcribed cagB gene. Primer extension analyses identified a single 5′ end of the cagA mRNA, while two initiation sites were mapped in the case of the cagB mRNA. The promoters deduced upstream of these start points of transcription contained conserved −10 regions but no −35 regions with respect to the Escherichia coliσ70 consensus sequence. Nevertheless, they could be activated in E. coli and in vitro by purified E. coli RNA polymerase. Deletion analyses indicated that the cagA and cagB genes are transcribed by overlapping promoters and that full activation requires sequences up to −70 and −96 respectively. Instead, basal transcription is likely to be mediated by −10 extended promoter‐like sequences. RNA polymerase is able to bind the −40 to −60 region of the cagA promoter, and its binding is mediated by the α‐subunit. This region resembles the UP elements of prokaryotic promoters in location, sequence and mechanism of interaction with the RNA polymerase. We discuss the features of these promoters and propose that they could represent a class of minimum promoters, which ensures a basic level of transcription, while full activation requires regulatory elements or a defined promoter context.
Global analysis of small RNA and mRNA targets of Hfq
Authors: Zhang Aixia , Wassarman Karen M. , Rosenow Carsten , Tjaden Brian C. , Storz Gisela , Gottesman Susan
Published: November 2003

Hfq, a bacterial member of the Sm family of RNA‐binding proteins, is required for the action of many small regulatory RNAs that act by basepairing with target mRNAs. Hfq binds this family of small RNAs efficiently. We have used co‐immunoprecipitation with Hfq and direct detection of the bound RNAs on genomic microarrays to identify members of this small RNA family. This approach was extremely sensitive; even Hfq‐binding small RNAs expressed at low levels were readily detected. At least 15 of 46 known small RNAs in E. coli interact with Hfq. In addition, high signals in other intergenic regions suggested up to 20 previously unidentified small RNAs bind Hfq; five were confirmed by Northern analysis. Strong signals within genes and operons also were detected, some of which correspond to known Hfq targets. Within the argX‐hisR‐leuT‐proM operon, Hfq appears to compete with RNase E and modulate RNA processing and degradation. Thus Hfq immunoprecipitation followed by microarray analysis is a highly effective method for detecting a major class of small RNAs as well as identifying new Hfq functions.
Identification and metabolic role of the mitochondrial aspartate‐glutamate transporter in Saccharomyces cerevisiae
Authors: Cavero S. , Vozza A. , Del Arco A. , Palmieri L. , Villa A. , Blanco E. , Runswick M. J. , Walker J. E. , Cerdán S. , Palmieri F. , Satrústegui J.
Published: November 2003

The malate‐aspartate NADH shuttle in mammalian cells requires the activity of the mitochondrial aspartate‐glutamate carrier (AGC). Recently, we identified in man two AGC isoforms, aralar1 and citrin, which are regulated by calcium on the external face of the inner mitochondrial membrane. We have now identified Agc1p as the yeast counterpart of the human AGC. The corresponding gene was overexpressed in bacteria and yeast mitochondria, and the protein was reconstituted in liposomes where it was identified as an aspartate‐glutamate transporter from its transport properties. Furthermore, yeast cells lacking Agc1p were unable to grow on acetate and oleic acid, and had reduced levels of valine, ornithine and citrulline; in contrast they grew on ethanol. Expression of the human AGC isoforms can replace the function of Agc1p. However, unlike its human orthologues, yeast Agc1p catalyses both aspartate‐glutamate exchange and substrate uniport activities. We conclude that Agc1p performs two metabolic roles in Saccharomyces cerevisiae. On the one hand, it functions as a uniporter to supply the mitochondria with glutamate for nitrogen metabolism and ornithine synthesis. On the other, the Agc1p, as an aspartate‐glutamate exchanger, plays a role within the malate‐aspartate NADH shuttle which is critical for the growth of yeast on acetate and fatty acids as carbon sources. These results provide strong evidence of the existence of a malate‐aspartate NADH shuttle in yeast.
ParG, a protein required for active partition of bacterial plasmids, has a dimeric ribbon–helix–helix structure
Authors: Golovanov Alexander P. , Barillà Daniela , Golovanova Marina , Hayes Finbarr , Lian Lu‐Yun
Published: November 2003

The ParG protein (8.6 kDa) is an essential component of the DNA partition complex of multidrug resistance plasmid TP228. ParG is a dimer in solution, interacts with DNA sequences upstream of the parFG genes and also with the ParF partition protein both in the absence and presence of target DNA. Here, the solution nuclear magnetic resonance structure of ParG is reported. The ParG dimer is composed of a folded domain formed by two closely intertwined C‐terminal parts (residues 33–76), and two highly mobile tails consisting of N‐terminal regions (residues 1–32). The folded part of ParG has the ribbon–helix–helix (RHH) architecture similar to that of the Arc/MetJ superfamily of DNA‐binding transcriptional repressors, although the primary sequence similarity is very low. ParG interacts with DNA predominantly via its folded domain; this interaction is coupled with ParG oligomerization. The dimeric RHH structure of ParG suggests that it binds to DNA by inserting the double‐stranded β‐sheet into the major groove of DNA, in a manner similar to transcriptional repressors from the Arc/MetJ superfamily, and that ParG can function as a transcriptional repressor itself. A new classification of proteins belonging to the Arc/MetJ superfamily and ParG homologues is proposed, based on the location of a conserved positively charged residue at either the beginning or at the end of the β‐strand which forms part of the DNA recognition motif.
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

In 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

Pyrimethamine 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

The 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

In 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

In 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

The 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

RNase 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

Type 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

The 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

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

Pili 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

The 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.
Phylogenetic relationships among Flavescence dorée strains and related phytoplasmas determined by heteroduplex mobility assay and sequence of ribosomal and Nonribosomal DNA
Authors: Angelini E. , Negrisolo E. , Clair D. , Borgo M. , Boudon‐Padieu E.
Published: November 2003

Heteroduplex mobility assay (HMA) and DNA sequencing were performed on Flavescence dorée (FD) phytoplasma strains and related phytoplasmas belonging to the elm yellows group. Part of the ribosomal RNA gene operon and a nonribosomal DNA region were utilized for phylogenetic analyses. Two FD strains, FD92 and FD‐D, detected in France and Italy, respectively, were identical in both DNA fragments, confirming previous results. Other FD strains were all very similar and most closely resembled ALY, an Italian alder phytoplasma. Phytoplasmas associated with German Palatinate grapevine yellows were shown to form a distinct subcluster, also different from the elm yellows phytoplasma subcluster. Strain disparities revealed by HMA and sequence data were mostly in agreement, highlighting the utility of HMA in differentiation and classification of phytoplasmas belonging to the same ribosomal RNA group.
Diversity of the coat protein‐coding region among Ilarvirus isolates infecting hop in Australia
Authors: Crowle D. R. , Pethybridge S. J. , Leggett G. W. , Sherriff L. J. , Wilson C. R.
Published: November 2003

Coat protein (CP) sequences of 17 Ilarvirus isolates were obtained from hops at three farms in Tasmania, Australia. Phylogenetic analysis of these sequences and additional database sequences indicated several Apple mosaic virus (ApMV) isolate clusters distinct from Prunus necrotic ringspot virus (PNRSV): one containing isolates from apple; one containing a single isolate from almond; a third containing Australian hop isolates of the ‘apple’ serotype and a German isolate of unknown origin; and a fourth containing Australian hop isolates of the ‘intermediate’ serotype. Isolates from hop, pear and prune from the Czech Republic either formed a fifth grouping, or were divergent members of the ‘intermediate’ serotype group. Deduced amino acid (aa) residue differences between the coat proteins of the two hop isolate serotype groups were highlighted as possible regions of serological differentiation. No evidence for coinfection of plants with both serotypes was found. Tests of ApMV‐infected hop buds using the Shirofugen flowering cherry assay revealed a possible differentiation of the two strains based on hypersensitivity. Because of serological similarities to PNRSV, these viruses have commonly been reported as strains of PNRSV. However, this study shows ilarviruses from Australian hops are strains of ApMV, but distinct from those infecting Malus spp.