bluuebunny/crossref_metadata_2025 huggingface dataset:
Predictors of refill non‐adherence in patients with heart failure
Authors: George Johnson , Shalansky Stephen J.
Published: October 2006

What is already known about this subject

• Non‐adherence to recommended treatment is common in patients with heart failure and is associated with poor outcomes.

• Personal beliefs as well as experiences with medications and illness could influence medication use.

What this study adds

• Perception regarding barrier to medication use was a stronger predictor of non‐adherence than demographic or clinical variables.

• Patients who were non‐adherent to nonpharmacological management of heart failure were more likely to be non‐adherent to their medications.

• Regimen complexity should not be considered in isolation when strategies for addressing adherence issues are designed.

Aim

To identify the health beliefs and patient characteristics associated with medication non‐adherence in patients attending a heart failure outpatient clinic.

Methods

A survey was administered to 350 consenting clinic patients. Questions focused on relevant demographic and clinical characteristics, the Health Belief Model, the Beliefs About Medicines Questionnaire and the Multidimensional Health Locus of Control. Multivariate logistic regression was used to identify independent predictors of refill non‐adherence (<90%).

Results

Refill non‐adherence was found in 77 (22%) participants. Being a smoker [odds ratio (OR) 2.4, 95% confidence interval (CI) 1.0, 5.8, P = 0.045], two or fewer medication administration times (OR 2.4, 95% CI 1.2, 4.6, P = 0.01), and positive response to ‘Have you changed your daily routine to accommodate your heart failure medication schedule’ (OR 2.4, 95% CI 1.2, 4.5, P = 0.01) were the independent predictors of refill non‐adherence.

Conclusion

Perceptions regarding barriers to medication taking and fewer administration times could result in medication non‐adherence in congestive heart failure patients. Medication regimens should be designed after accounting for patients' existing routines.
SHOX mutations in idiopathic short stature and Leri‐Weill dyschondrosteosis: frequency and phenotypic variability
Authors: Jorge Alexander A. L. , Souza Silvia C. , Nishi Miriam Y. , Billerbeck Ana E. , Libório Débora C. C. , Kim Chong A. , Arnhold Ivo J. P. , Mendonca Berenice B.
Published: October 2006

Objective  The frequency of SHOX mutations in children with idiopathic short stature (ISS) has been found to be variable. We analysed the SHOX gene in children with ISS and Leri‐Weill dyschondrosteosis (LWD) and evaluated the phenotypic variability in patients harbouring SHOX mutations.

Patients  Sixty‐three ISS, nine LWD children and 21 affected relatives.

Methods  SHOX gene deletion was evaluated by fluorescence in situ hybridization (FISH), Southern blotting and segregation study of polymorphic marker. Point mutations were assessed by direct DNA sequencing.

Results  None of the ISS patients presented SHOX deletions, but two (3·2%) presented heterozygous point mutations, including the novel R147H mutation. However, when ISS patients were selected by sitting height : height ratio (SH/H) for age > 2 SD, mutation frequency detection increased to 22%. Eight (89%) LWD patients had SHOX deletions, but none had point mutations. Analysis of the other relatives in the families carrying SHOX mutations identified 14 children and 17 adult patients. A broad phenotypic variability was observed in all families regarding short stature severity and Madelung deformities. However, the presence of disproportional height, assessed by SH/H, was observed in all children and 82% of adult patients, being the most common feature in our patients with SHOX mutations.

Conclusion  Patients with SHOX mutations present a broad phenotypic variability. SHOX mutations are very frequent in LWD (89%), in opposition to ISS (3·2%) in our cohort. The use of SH/H SDS as a selection criterion increases the frequency of SHOX mutation detection to 22% and should be used for selecting ISS children to undergo SHOX mutation molecular studies.
Theoretical model of coping among relatives of patients in intensive care units: a simultaneous concept analysis
Authors: Johansson Ingrid , Hildingh Cathrine , Wenneberg Stig , Fridlund Bengt , Ahlström Gerd
Published: October 2006

Aim.  This paper reports the development of a theoretical model of relatives’ coping approaches during the patient's intensive care unit stay and subsequent recovery at home by performing an analysis of concepts generated from two empirically grounded, theoretical studies in this area.

Background.  When supporting relatives of intensive care unit patients, it is important that nurses have access to evidence‐based knowledge of relatives’ coping approaches during the period of illness and recovery.

Method.  Simultaneous concept analysis was used to refine and combine multiple coping concepts into a theoretical model of coping. The concepts were generated in two previous empirical studies of relatives’ coping approaches during mechanically ventilated patients’ intensive care unit stays and recovery at home.

Findings.  The theoretical model was developed in 2004–2005 and illustrates the effectiveness of different coping approaches in relation to each other and to social support. Definitions summarizing each coping approach and containing the knowledge gained through the simultaneous concept analysis method were also formulated.

Conclusion.  This middle‐range theory of relatives’ coping approaches may make a valuable contribution to international intensive care unit nursing practice, especially as it is based on empirical studies and may therefore serve as a basis for the development of future clinical guidelines. However, the theoretical model needs to be empirically validated before it can be used.
Development of a framework for person‐centred nursing
Authors: McCormack Brendan , McCance Tanya V.
Published: October 2006

Aim.  This paper presents the development and content of a person‐centred nursing framework.

Background and rationale.  Person‐centred is a widely used concept in nursing and health care generally, and a range of literature articulates key components of person‐centred nursing. This evidence base highlights the links between this approach and previous work on therapeutic caring.

Methods.  The framework was developed through an iterative process and involved a series of systematic steps to combine two existing conceptual frameworks derived from empirical studies. The process included the mapping of original conceptual frameworks against the person‐centred nursing and caring literature, critical dialogue to develop a combined framework, and focus groups with practitioners and co‐researchers in a larger person‐centred nursing development and research project to test its face validity.

Findings.  The person‐centred nursing framework comprises four constructs –prerequisites, which focus on the attributes of the nurse; the care environment, which focuses on the context in which care is delivered; person‐centred processes, which focus on delivering care through a range of activities; and expected outcomes, which are the results of effective person‐centred nursing. The relationship between the constructs suggests that, to deliver person‐centred outcomes, account must be taken of the prerequisites and the care environment that are necessary for providing effective care through the care processes.

Conclusion.  The framework described here has been tested in a development and research project in an acute hospital setting. Whilst there is an increasing empirical base for person‐centred nursing, as yet little research has been undertaken to determine its outcomes for patients and nurses. The framework developed can be described as a mid‐range theory. Further testing of the framework through empirical research is required to establish its utility for nursing practice and research.
Control of functional mRNA stability in bacteria: multiple mechanisms of nucleolytic and non‐nucleolytic inactivation
Authors: Petersen C.
Published: October 2006

Messenger RNA in bacteria may be inactivated by several parallel mechanisms acting independently on different target sites. For any species of mRNA the overall rate of inactivation is determined by the sum of the contributions from the different mechanisms. Transcripts may be inactivated directly by endonucleolytic attack or by processive nucleolytic degradation, which may proceed in the 3′–5′ direction and probably also in the 5′‐3′ direction. Moreover, the functional lifetime of many mRNAs may be determined by processes that are not nucleolytic, such as the binding of translational repressors or the formation of secondary structures which prevent initiation of translation. These non‐nucleolytic processes may also determine the chemical stability as chemical degradation frequently appears to be closely coupled to functional inactivation. The relative importance of the different mechanisms in the inactivation of bulk cellular mRNA, as well as the general prospects for engineering of stable mRNAs are discussed.
D‐E‐A‐D protein family of putative RNA helicases
Authors: Schmid S. R. , Linder P.
Published: October 2006

RNA metabolism plays a central role in cell growth. It is essential to regulate RNA synthesis, processing, stability and degradation. Conformational changes in RNA are key elements in regulating cellular processes. Recently, an increasing number of putative RNA helicases from different organisms ranging from Escherichia coli to humans and viruses have been identified. They are Involved in diverse cellular functions such as RNA splicing, ribosome assembly, initiation of translation, spermatogenesis, embryogenesis, and cell growth and division. Based on sequence homologies these proteins were grouped in a family, the D‐E‐A‐D box protein family (D‐E‐A‐D = Asp‐Glu‐Ala‐Asp). Some of the better characterized members have been shown to possess ATP‐binding and hydrolysing activities as well as ATP‐dependent RNA helicase activities. Most of the genes encoding such proteins have been isolated from yeast, on which we will focus in this review. From sequence data, three of the members form a subfamily, the D‐E‐A‐H subfamily.
CooB is required for assembly but not transport of CS1 pilin
Authors: Scott J. R. , Wakefield J. C. , Russell P. W. , Orndorff P. E. , Froehlich B. J.
Published: October 2006

CS1 pili are filamentous proteinaceous appendages found on many enterotoxigenic Escherichia coli (ETEC) strains isolated from human diarrhoeal disease. They are thought to effect colonization of the upper intestine by facilitating binding to human ileal epithelial cells. We have identified a gene, cooB, which lies directly upstream of cooA, the gene that encodes the major structural CS1 protein. When translated in vitro, the protein product of cooB migrates in sodium dodecyl sulphate/polyacrylamide gel with an apparent molecular mass of 26 kDa, which is consistent with that predicted from its DNA sequence. We constructed a mutant allele (cooB‐1) by insertion of the omega fragment, which inhibits transcription and translation, into the cooB gene in vitro. In a derivative of an ETEC strain with the cooB‐1 mutation (JEF100) and a plasmid that encodes Rns (pEU2030), the positive regulator required for CS1 expression, no cooB and a greatly reduced level of cooA product was detectable in total cell extracts. The reduction of cooA in this strain appears to result from polarity of the cooB mutation because introduction of the wild‐type cooA gene in trans causes production of CooA protein, which is found in cell pellet extracts, in extracts containing only surface proteins and in the culture supernatant. Therefore, in the absence of CooB, CooA is stable and it is transported through both inner and outer membranes. However, the cooB‐1 strain with cooA in trans does not cause haemagglutination of bovine erythrocytes (the model system used to assay adherence mediated by coli surface antigen 1 (CS1) pili). Electron microscopy reveals that there are no pili present on the cell surface or in the culture medium in which this strain was grown. Thus, although it is not required for stability or transport of the major CS1 pilin protein, CooB is needed for assembly of CooA into pili. The relationship of these gene products to those of other pili is discussed.
Cloning of a yeast gene coding for the glutamate synthase small subunit (GUS2) by complementation of Saccharomyces cerevisiae and Escherichia coli glutamate auxotrophs
Authors: Gonzàlez A. , Membrillo‐Hernández J. , Olivera H. , Aranda C. , Macino G. , Ballario P.
Published: October 2006

A Saccharomyces cerevisiae glutamate auxotroph, lacking NADP‐glutamate dehydrogenase (NADP‐GDH) and glutamate synthase (GOGAT) activities, was complemented with a yeast genomic library. Clones were obtained which still lacked NADP‐GDH but showed GOGAT activity. Northern analysis revealed that the DNA fragment present in the complementing plasmids coded for a 1.5kb mRNA. Since the only GOGAT enzyme so far purified from S. cerevisiae is made up of a small and a large subunit, the size of the mRNA suggested that the cloned DNA fragment could code for the GOGAT small subunit. Plasmids were purified and used to transform Escherichia coli glutamate auxotrophs. Transformants were only recovered when the recipient strain was an E. coli GDH‐less mutant lacking the small GOGAT subunit. These data show that we have cloned the structural gene coding for the yeast small subunit (GUS2). Evidence is also presented indicating that the GOGAT enzyme which is synthesized in the E. coli transformants is a hybrid comprising the large E. coli subunit and the small S. cerevisiae subunit.
Cloning of an autonomously replicating sequence (ars) from the Bacillus subtilis chromosome
Authors: Moriya S. , Atlung T. , Hansen F. G. , Yoshikawa H. , Ogasawara N.
Published: October 2006

Cloning of an autonomously replicating sequence (ars) from the origin region of Bacillus subtilis was previously unsuccessful because of the strong incompatibility exerted by sequences located within the oriC region. Using an ars searching vector which would be selective for drug resistance even at one copy per cell, and by cloning large fragments covering as much as possible of the oriC region, we have succeeded in isolating ars fragments from the origin region of the chromosome. The minimum essential fragment contains two DnaA‐box regions (non‐translatable regions containing multiple repeats of DnaA‐box) separated by the dnaA gene. Neither one of the DnaA‐box regions by itself showed ars activity. When constructed as oriC plasmids, the dnaA coding region could be removed without affecting ars activity. The minimum distance between the two DnaA‐box regions obtained so far is 274 bp. The copy number of the oriC plasmid is estimated as one per replicating chromosome. These plasmids are unstable and tend to be lost or integrated into chromosome.
Zinc and iron regulate translation of the gene encoding Pseudomonas aeruginosa elastase
Authors: Brumlik M. J. , Storey D. G.
Published: October 2006

A lasB–lacZ translational fusion (pTS400) was used to examine expression of the elastase gene (lasB) in Pseudomonas aeruginosa strain PAO1. Expression from the lasB–lacZ fusion was enhanced when PAO1(pTS400) was grown in a defined medium containing elevated levels of zinc (6.0μg ml‐1). Transcript accumulation studies on PAO1(pTS400) and PAO1 showed that the addition of zinc had a slight negative effect on lasB transcription. These results indicated that zinc regulates the expression of elastase at the translational level. A comparison between zinc regulation and iron regulation was also made. Iron has a negative effect on lasB–lacZ expression. When PAO1(pTS400) was grown in a defined medium with a low iron content (0.1 μg ml‐1) the bacteria still responded to zinc. The independent effects of low iron and high zinc concentrations suggest separate control mechanisms for the two factors. Transcript accumulation studies on PA01 and PAO1(pTS400) indicated that early in the growth curve iron did not influence transcription of tasB or lasB–lacZ. Later in the growth curve a slight increase in lasB–lacZ transcription was observed only in PAO1(pTS400) grown in low iron. These results suggest that the iron regulation of lasB occurs predominantly at the transtational level. Finally, when PAO1(pTS400) was grown in a complex peptone‐based medium, a high level of transcript accumulation accounted for elastase expression. Alterations of iron and zinc concentrations of this medium did not affect the expression of elastase. These results suggest that there may be additional environmental cues regulating lasB transcription.
Retron‐Ec107 is inserted into the Escherichia coli genome by replacing a palindromic 34bp intergenic sequence
Authors: Herzer P. J. , Inouye S. , Inouye M.
Published: October 2006

Some natural isolates of Escherichia coli have been shown to produce a unique branched RNA‐linked single‐stranded DNA called msDNA. These bacteria contain a retro‐element called retron consisting of the msr–msd region and the gene for reverse transcriptase (RT). All three E. coli retrons characterized to date have been shown to be integrated into a prophage or to be associated with phage‐related genes. In this report, we identified a new msDNA from an E. coli wild strain. Using the msDNA as a probe, the retron for the msDNA was cloned and its DNA sequence was determined. The retron was found to consist of a 1.3 kb DNA fragment, making it the smallest retron isolated to date. The msDNA produced from the retron consists of a 107 base single‐stranded DNA, which is considered to be branched out from the 18th G residue of a 75‐base RNA molecule by a 2′,5′‐phosphodiester linkage. Thus, the msDNA and the retron were designated msDNA‐Ec107 and retron‐Ec107, respectively. Most significantly, retron‐Ec107 was inserted into the E. coli genome by replacing a 34bp intergenic sequence between the pyrE and ttk genes located at 82 min on the E. coli chromosome. Interestingly, the retron contains palindromic structures at both ends and the E. Coli 34bp intergenic sequence also contains a 10bp inverted repeat structure. These palindromic structures might have played a role in the integration of retron‐Ec107 into the E. coli genome.
Efficient repression by a heterodimeric repressor in Escherichia coli
Authors: Webster C. , Merryweather A. , Brammar W.
Published: October 2006

Previous studies with purified variants of the 434 repressor having different operator‐binding specificities have demonstrated the interactions of a heterodimeric repressor with a hybrid operator site. The present study investigates the interactions between the 434 repressor and its operator site. The optimum 434 operator half‐site is used with a P22 operator half‐site to create a hybrid 434/P22 operator. We show that this hybrid operator can be efficiently bound by a heterodimeric repressor, consisting of one wild‐type 434 repressor monomer and one 434 repressor monomer with the binding specificity of the P22 repressor, to bring about repression in Escherichia coli.
Role of lipid modification on a starch‐debranching enzyme, Klebsieila pullulanase: comparison of properties of lipid‐modified and unmodified pullulanases
Authors: Yamashita M. , Nakagawa A. , Katsuragi N. , Murooka Y.
Published: October 2006

Klebsiella pullulanase is a lipoprotein synthesized as a precursor with a signal peptide, which is processed by lipoprotein signal peptidase. To clarify the role of lipid modification of pullulanase, we purified lipid‐modified wild‐type and the unmodified (mutant) pullulanases and compared their properties. The Km and Vmax values of both pullulanases for pullulan were the same. The optimal pH and temperature, the stabilities over pH and temperature ranges, the specificity of substrates, and the patterns of inhibition of the lipid‐modified and unmodified pullulanases were also the same. However, we found that the wild‐type pullulanase formed trimers whereas the unmodified enzyme did not, and that the migrations of the two enzymes on sodium dodecyl sulphate/electrophoresis were different when the samples were applied on the gel without heating. The results presented in this paper and in previous work show that the correct processing and translocation of pullulanase in K. aerogenes require modification of lipid. However, the enzymatic properties and physical stabilities of pullulanase were not affected by the lipid modification.
Role of the transcriptional activator, VirF, and temperature in the expression of the pYV plasmid genes of Yersinia enterocolitica
Authors: de Rouvroit C. Lambert , Sluiters C. , Cornelis G. R.
Published: October 2006

Upon incubation at 37°C in the absence of Ca2+ ions, pathogenic yersiniae release high amounts of pYV plasmid‐encoded proteins called Yops, involved in pathogenesis. Yersinia enterocolitica also express two outer membrane proteins, an adhesin called YadA and a lipoprotein called YIpA. The production of the Yops is co‐ordinately regulated by a 20kb region of the plasmid referred to as the ‘Ca2+ dependence region’ and containing at least four loci called virA, virB, virC, and virF. The 8.5kb virC region, involved in the specific transport of the Yops, is a single operon containing 13 open reading frames called yscA to yscM. Gene virF encodes a key transcriptional activator of the yop, yadA and ylpA genes. It is only transcribed at 37°C and its expression is modulated by a chromosome‐encoded histone‐like protein called YmoA. We show here that virF also controls the virC operon. By contrast, VirF is not essential for the induction of virA and virB. The VirF protein binds specifically to yop promoters. In particular, it protects the region spanning nucleotides ‐64 to ‐34 of yopH. In order to analyse the role of temperature in the induction of the yop regulon, we constructed Y. enterocolitica strains expressing virF from the tac promoter. In spite of the fact that virF was transcribed at 25°C, neither the Yops nor YadA were expressed at that temperature. This poor response to VirF at 25°C was at least partially due to a weak and slow transcription of the genes controlled by virF. Surprisingly, when cloned on pACYC184, gene yadA was expressed even in absence of VirF, but remained thermodependent. Hence temperature and virF are both required for the induction of the yop regulon. Among other possible roles, temperature could modify the structure of either the activator itself or the yop promoter. The fact that VirF binds in vitro to yop promoters at 25°C rules out the first hypothesis. In order to test the second hypothesis, we studied, in vivo, the activity of the yopH promoter in ymoA mutants. The yopH promoter became active in the absence of VirF, indicating that yop promoter activity depends upon chromatin structure. We conclude from these two observations that, in vivo, temperature is required to modify the DNA structure of the yop promoters in order to allow the action of the transcriptional activator.
Cloning and characterization of the eae gene of enterohaemorrhagic Escherichia coli O157:H7
Authors: Yu J. , Kaper J. B.
Published: October 2006

The eae (Escherichia collattaching and effacing) gene from enteropathogenic Escherichia coli (EPEC) was previously shown to be essential for production of the ‘attaching and effacing’ histopathology characteristic of EPEC infections (Jerse et al., 1990). We have now cloned the eae gene from enterohaemorrhagic E. coli (EHEC) which, in addition to producing Shiga‐like cytotoxins, also produces the attaching and effacing effect. The sequence homology between the EPEC and EHEC sequences was 86% and 83% at the nucleotide and amino acid levels, respectively. The predicted amino acid sequence of the EHEC eae gene shared 31% identity and 51 % similarity with invasin of Yersinia pseudotuberculosis. Alignment of the EPEC and EHEC Eae proteins and the Y. pseudotuberculosis and Y. enterocoltica invasins shows striking regions of identity with the greatest divergence at the C‐terminal end, the putative receptor‐binding portion of invasin.
DNA loops: structural and functional properties of scaffold‐attached regions
Authors: Roberge Michel , Gasser Susan M.
Published: October 2006

The long DNA molecules of eukaryotic genomes appear to be organized into large loops formed by the binding of dispersed DNA sequences to non‐histone proteins. This partitioning of DNA into topologically constrained units constitutes one of the highest orders of DNA packing in chromosomes. DNA loops are likely to define functional units as well as topological domains, contributing to the regulation of gene expression and DNA replication. This review presents recent work on the properties of the DNA sequences and proteins thought to be involved in loop formation, and on their possible significance for replication and transcription.
Control of bacterial DNA supercoiling
Authors: Drlica Karl
Published: October 2006

Two DNA topoisomerases control the level of negative supercoiling in bacterial cells. DNA gyrase introduces supercoils, and DNA topoisomerase I prevents super‐coiling from reaching unacceptably high levels. Perturbations of supercoiling are corrected by the substrate preferences of these topoisomerases with respect to DNA topology and by changes in expression of the genes encoding the enzymes. However, super‐coiling changes when the growth environment is altered in ways that also affect cellular energetics. The ratio of [ATP] to [ADP], to which gyrase is sensitive, may be involved in the response of supercoiling to growth conditions. Inside cells, supercoiling is partitioned into two components, superhelical tension and restrained supercoils. Shifts in superhelical tension elicited by nicking or by salt shock do not rapidly change the level of restrained supercoiling. However, a steady‐state change in supercoiling caused by mutation of topA does alter both tension and restrained supercoils. This communication between the two compartments may play a role in the control of supercoiling.
Co‐ordinate expression of virulence genes by ToxR in Vibrio cholerae
Authors: DiRita Victor J.
Published: October 2006

Evolution of complex regulatory pathways that control virulence factor expression in pathogenic bacteria indicates the importance to these organisms of being able to distinguish time and place. In the human intestinal pathogen Vibrio cholerae, control over many virulence genes identified to date is the responsibility of the ToxR protein. ToxR, in conjunction with a second regulatory protein called ToxS, directly activates the genes encoding the cholera toxin; other ToxR regulated genes are not activated directly by ToxR. For some of these genes, ToxR manifests its control through another activator called ToxT. Expression of toxT, which encodes a member of the AraC family of bacterial transcriptional activators, is ToxR dependent and is modulated by in vitro growth conditions that modulate expression of the ToxR virulence regulon. Thus, as in other regulatory circuits, co‐ordinate expression of several genes in V. cholerae results from the activity of a cascading system of regulatory factors.
The filA (rpoF) gene of Pseudomonas aeruginosa encodes an alternative sigma factor required for flagellin synthesis
Authors: Starnbach M. N. , Lory S.
Published: October 2006

In order to better understand the regulation of Pseudomonas aeruginosa flagellin expression we cloned the sigma factor of RNA polymerase used to transcribe the flagellin gene. It is a member of the σ28 class of alternative sigma factors described in several bacterial genera. Using the published sequence of the filA gene encoding the σ28 from Salmonella typhimurium, we designed two oligonucleotides and, using the polymerase chain reaction, isolated the fliA gene from S. typhimurium chromosomal DNA. This heterologous probe was used in the DNA blot analysis of restriction digests of P. aeruginosa DNA. A 1.7 kb SalI‐EcoRI fragment reacted with the probe and this fragment was cloned into the pBluescript vectors. The P. aeruginosa fliA gene was able to complement the motility defect of an Escherichia coli fliA mutant, but only when transcription was driven from the vector promoter. Insertional inactivation of the fliA gene with a gentamicin gene cassette rendered P. aeruginosa non‐motile and unable to express the flagellin gene. The 1.7 kb cloned fragment was sequenced and shown to contain the entire fliA gene. P. aeruginosa FliA shares 67% amino acid similarity with the homologous S. typhimurium sequence. Transcriptional analysis of the fliA gene showed that its expression was not dependent on RpoN, a sigma factor shown also to be required for flagellin synthesis. A reading frame downstream of fliA was found to encode the P. aeruginosa homologue of the enterobacterial cheY gene.
The adsorption protein of phage IKe. Localization by deletion mutagenesis of domains involved in infectivity
Authors: Endemann H. , Bross P. , Rasched I.
Published: October 2006

We constructed a set of plasmid‐encoded internal deletion mutants within the gene for the adsorption protein (g3p) of phage IKe. All mutant proteins still contain the signal and membrane anchor sequence, as those are known to be indispensable for proper localization and hence assembly of the g3p into phage. These various deletions comprise ail internal parts of the protein and are properly incorporated into phage, which remarkably shows that signal and anchor sequence are sufficient for incorporation of g3p.

The data furthermore reveal that two separate sections within the IKe g3p are essential for infection: one amino‐terminal, preceding the glycine‐rich stretch, and the other carboxy‐terminal. We conclude that this latter domain is involved in penetration because mutants tacking it are not infectious, but still bind to the receptor. The amino‐terminal region, essential for infection, bears the receptor‐recognizing domain and a sequence homologous to the penetration domain of the evolutionary related Ff phages, which is probably also involved in penetration of phage IKe.

The prominent glycine‐rich stretch of the IKe g3p is not essential for infection but significantly promotes it.
Suppression of the ndv mutant phenotype of Rhizobium meliloti by cloned exo genes
Authors: Nagpal Punita , Khanuja Suman P. S. , Stanfield Sharon W.
Published: October 2006

The ndv A and ndvB genes of Rhizobium meliloti are involved in the export and synthesis, respectively, of the small cyclic polysaccharide β(1,2)glucan. We have previously shown that spontaneous symbiotic pseudorevertants of ndv mutants do not produce periplasmic β(1,2)glucan. Here we show that the pseudorevertants also do not produce extracellular β(1,2)glucan, but do show alterations in the amount of the major acidic exopolysaccharide produced. This exopolysaccharide is not detectably different from that produced by the wild type or by the ndv mutants. A cosmid which suppresses the symbiotic defect of both ndvA and ndvB mutants was isolated from a gene bank prepared from DNA of an ndvA pseudo‐revertant. This cosmid contains a number of exo genes, including exoH and exoF. Subcloning and Tn5 mutagenesis were used to show that the widely separated exoH and exoF genes are both involved in suppression of the ndv mutant phenotype and that the 3.5 kb DNA fragment which contains the exoH gene does not carry the mutation responsible for second site suppression.
Kex2‐dependent processing of yeast K1 killer preprotoxin includes cleavage at ProArg‐44
Authors: Zhu Yun‐Song , Zhang Xia‐Ying , Cartwright Charles P. , Tipper Donald J.
Published: October 2006

The K1 killer toxin of Saccharomyces cerevisiae consists of 103‐ and 83‐residue α and β components whose derivation, from a 316‐residue precursor preprotoxin, requires processing at the αN‐terminus (after ProArg‐44), the αC‐terminus (after ArgArg‐149) and at the βN‐terminus (after LysArg–233). These processing events occur after translocation to the Golgi and have been investigated using β‐lactamase fusions. Signal peptidase cleavage of the precursor, predicted to occur after Ala‐26, was confirmed by N‐terminal sequence analysis of Ala‐34 and IIe‐52 fusions. Cleavage at all of the other predicted processing sites, including ProArg‐44, is dependent on activity of the Kex2 protease. A fourth Kex2‐dependent cleavage occurs at LysArg‐188. Implications for the specificity of Kex2 cleavage and preprotoxin processing are discussed.
A gene near the plasmid pSa origin of replication encodes a nuclease
Authors: Closed S. M. , Kado C. I.
Published: October 2006

We have cloned and sequenced a gene (nuc) from the IncW plasmid pSa which shows amino acid sequence similarity to staphylococcal nuclease (EC 3.1.4.7) and to the parB locus of plasmid RP4. The 525bp open reading frame encodes a 174‐amino‐acid potential polypeptide of 19.7kDa. Expression of the gene was confirmed using an in vitro transcription‐translation assay which produced a protein of identical size. Nuclease activity was demonstrated using DNA as the substrate in toluidine blue‐DNA agar plates. The deduced amino acid sequence revealed a signal sequence, and TnphoA insertion within the open reading frame indicated that a portion of the protein is transported across the bacterial cell membrane.
Four homologous domains in the primary structure of GrsB are related to domains in a superfamily of adenylate‐forming enzymes
Authors: Turgay K. , Krause M. , Marahiel M. A.
Published: October 2006

The entire nucleotide sequence of the Bacillus brevis grsB gene encoding the gramicidin S synthetase 2, which activates and condenses the four amino acids proline, valine, ornithine and leucine has been determined. The gene contains an open reading frame of 13359bp which encodes a protein of 4453 amino acids with a predicted Mr of 510287. The gene is located within the gramicidin S biosynthetic operon, also containing the genes grsT and grsA, whose nucleotide sequences have been determined previously. Within the GrsB amino acid sequence four conserved and repeated domains of about 600 amino acids (45–50% identity) have been identified. The four domains are separated by non‐homologous sequences of about 500 amino acids. The domains also share a high degree of similarity (20–70%) with eight peptide synthetases of bacterial and fungal origin as well as with conserved sequences of nine other adenylate‐forming enzymes of diverse origin. On the basis of sequence homology and functional similarities, we infer that those enzymes share a common evolutionary origin and present a phylogenetic tree for this superfamily of domain‐bearing enzymes.
Outer membrane protein P6 of Haemophilus influenzae binds to its own gene
Authors: Sikkema Daniel J. , Nelson M. Bud , Apicella Michael A. , Murphy Timothy F.
Published: October 2006

Outer membrane protein P6 is an important antigen expressed on the surface of all strains of Haemophilus influenzae. The predicted amino acid sequence of P6 contains a region of alpha helices that shares sequence identity with a family of helix‐turn‐helix DNA‐binding proteins. A search for sequence‐specific binding sites that resemble an operator region within the gene revealed a sequence with striking homology to the consensus operator sequence for lambda Cro and repressor. To test the hypothesis that P6 binds its own gene, purified P6 on nitrocellulose was probed with plasmid DNA containing the P6 gene. P6 bound the P6 gene in this Southwestern blot assay. To further test the observation, gel shift analysis was performed. Gel shift assays using a P6‐specific monoclonal antibody demonstrated that P6 in crude cell extracts binds to the region of the gene containing the putative binding site. Competition with a synthetic oligonucleo‐tide corresponding to the putative binding site inhibited binding of P6 to the P6 gene on nitrocellulose and in the gel shift assay. In addition, this oligonucleotide bound directly to P6 on nitrocellulose. Finally, DNase footprinting confirmed that P6 bound specifically to the same region of the P6 gene. These results indicate that P6 binds to a sequence‐specific site within its own gene, suggesting that P6 regulates its own expression. This represents the first example of a Gram‐negative outer membrane protein binding to its own gene and has potentially important implications as a mechanism for regulation of expression of outer membrane antigens.
A theoretical and experimental analysis of bacterial growth in the bladder
Authors: Gordon David M. , Riley Margaret A.
Published: October 2006

A mathematical model of human micturition dynamics and bacterial growth predicts the population growth rate required for a bladder infection to become established in the absence of adhesin‐mediated surface growth. Escherichia coli strains isolated from the urinary tract have significantly higher in vitro growth rates in urine than strains isolated from the intestinal flora. The results suggest that, for E. coli isolated from the urinary tract, adhesin‐mediated surface growth may not be required for infections to become established and persist. The growth‐rate differences observed between urinary tract and Intestinal isolates suggests that the ability to survive and efficiently utilize the resources available in urine is an important adaptation for E. coli inhabiting the urinary tract.
DNA in dormant spores of Bacillus species is in an A‐like conformation
Authors: Setlow Peter
Published: October 2006

The DNA in dormant spores of Bacillus species is associated with α/β‐type small, acid‐soluble proteins (SASP), which are double‐stranded DNA‐binding proteins whose amino acid sequence has been highly conserved in evolution. In vitro these proteins bind most strongly to DNA which readily adopts an A‐like conformation, as binding of α/β‐type SASP causes DNA to assume an A‐like conformation. As predicted by this conformational change in DNA, binding of α/β‐type SASP to relaxed but covalently closed plasmid DNA results in the introduction of a large number of negative supercoils. Associated with the conformational change in DNA brought about by α/β‐type SASP binding is a change in its photochemistry such that ultraviolet irradiation does not generate pyrimidine dimers, but rather a thyminyl‐thymine adduct termed spore photoproduct (SP). The latter two properties of DNA complexed with α/β‐type SASP in vitro are similar to those of DNA in dormant spores of Bacillus species in which: (i) plasmid DNA has a much higher number of negative supercoils than plasmid in growing cells; and (ii) ultraviolet irradiation produces SP and no pyrimidine dimers, while only pyrimidine dimers are formed in growing cells. During sporulation these changes in the properties of spore DNA take place in parallel with synthesis of α/β‐type SASP, and the magnitude of the changes is greatly reduced in mutants that make low amounts of these proteins. A straightforward interpretation of these data is that DNA in dormant spores of Bacillus species is in an A‐like conformation. DNA in an A‐like conformation is also present in growing cells with high α/β‐type SASP levels. However, the presence of this A‐like DNA is detrimental to cell growth.
Z‐DNA as a probe for localized supercoiling in vivo
Authors: Rahmouni A. Rachid
Published: October 2006

Biological processes such as transcription are expected to generate local variations in DNA super‐coiling. The existence of localized supercoiling was recently demonstrated in Escherichia coli by using the supercoil‐driven B‐to‐Z transition as a super‐helicity probe. This new methodology is described and its extension to other biological systems discussed.
Effects of template topology on RNA polymerase pausing during in vitro transcription of the Escherichia coli rrnB leader region
Authors: Krohn M. , Pardon B. , Wagner R.
Published: October 2006

Transcription elongation catalysed by DNA‐dependent RNA polymerase does not occur at a constant rate. Instead, during the transcription of many genes pausing occurs at defined template positions. Pausing is known to be influenced by the intracellular NTP concentration, the secondary structure of the growing transcript or by transcription factors like NusA. We have investigated the effects of the template topology of transcriptional pauses in the presence and absence on purified NusA protein. Taking advantage of a method for quantifying transcriptional pauses we have studied pausing behaviour during in vitro transcription of the early region of a plasmid‐encoded ribosomal RNA operon. Plasmid templates with different super‐helical densities (σ between +0.0017 and ‐0.055) were employed in transcription elongation assays. If linearized or relaxed templates are used, some of the characteristic pauses can no longer be detected. For the stronger pauses we could demonstrate a direct correlation between pause strength and the negative superhelical densities of the templates used. This correlation is observed regardless of whether or not pauses are dependent upon NusA. Changes in the average transcription elongation rate, caused by variations in the NTP concentration or the temperature, do not appear to have a comparable effect on transcription pausing. The results are consistent with the assumption that the template topology has a regulatory function in transcription elongation of rRNA genes in Escherichia coli.
Tumour necrosis factor alpha antibody protects against lethal meningococcaemia
Authors: Nassif X. , Mathison J. C. , Wolfson E. , Koziol J. A. , Ulevitch R. J. , Son M.
Published: October 2006

Tumour necrosis factor alpha (TNF‐α) has been shown to be the principal mediator of Gram‐negative bacterial endotoxin‐induced shock. Nevertheless, evidence suggests that TNF‐α plays a beneficial role in controlling bacterial infections when multiplication of the microorganism is required to kill the host. Using an infant rat model of Neisseria meningitidis infection, we found that blood TNF‐α concentration reaches a peak three hours after intraperitoneal injection of 3 × 106 bacteria. Thereafter, the level of TNF‐α decreased and was undetectable six to eight hours after infection. A correlation was observed between the magnitude of initial TNF‐α response and a fatal outcome. Pretreatment of the animals with polyclonal anti‐TNF antiserum significantly reduced mortality relative to animals pretreated with control serum. However, pretreatment of animals with anti‐TNF antibody did not alter the bacterial invasion of the cere‐brospinal fluid. Injection of heat‐killed bacteria did not cause death and induced lower TNF‐α levels than the same number of live bacteria. This excludes the possibility that the role of TNF‐α is to mediate a shock induced by the endotoxin component of the bacterial inoculum. These results indicate that TNF‐α has a deleterious effect in this model of bacteraemia. Identification of the critical factors that determine the action of TNF‐α during lethal bacteraemia will lead to a better understanding of these diseases and the development of appropriate therapeutic intervention.
SecB‐binding does not maintain the translocation‐competent state of prePhoE
Authors: de Cock Hans , Tommassen Jan
Published: October 2006

The rote of SecB protein in the export of the precursor of outer membrane protein PhoE and mutant forms of this precursor was studied in vitro. When synthesized in the absence of SecB, translocation‐competent prePhoE was observed post‐translationally, but addition of SecB was required for efficient translocation into inner membrane vesicles. The translocation competency of in vitro synthesized prePhoE diminished with a similar half‐life during incubations in the presence or absence of SecB. The loss of translocation competency of prePhoE, synthesized in the presence of SecB, was not due to dissociation of prePhoE–SecB complexes as could be demonstrated in co‐immunoprecipitation experiments with anti‐SecB serum. Apparently, SecB does not maintain the translocation‐competent conformation of prePhoE, but is mainly required for efficient targeting of this precursor to the export apparatus.
Regulation of the expression of the cell‐cycle gene ftsZ by DicF antisense RNA. Division does not require a fixed number of FtsZ molecules
Authors: Tétart Françoise , Bouché Jean‐Pierre
Published: October 2006

We show that the 53‐nucieotide RNA molecule encoded by gene dicF blocks cell division In Escherichia coli by inhibiting the translation of ftsZ mRNA. Such a role for dicF had been predicted on the basis of the complementarity of DicF RNA with the ribosome‐binding region of the ffsZ mRNA. An analysis of ftsZ expression at its chromosomal locus, and of an ftsZ–lacZ translational fusion controlled by promoters ftsZ1p and ftsZ2p only, indicates that ftsZ is not autoregulated. Partial inhibition of FtsZ synthesis leads to increased cell size. However, the number of FtsZ molecules per cell can be reduced threefold without affecting the division rate significantly. Our results suggest that septation is not triggered by a fixed number of newly synthesized FtsZ molecules per cell.
Genes and their organization in the replication origin region of the bacterial chromosome
Authors: Ogasawara Naotake , Yoshikawa Hiroshi
Published: October 2006

Genes and their organization are conserved in the replication origin region of the bacterial chromosome. To determine the extent of the conserved region in Gram‐positive and Gram‐negative bacteria, which diverged 1.2 billion years ago, we have further sequenced the region upstream from the dnaA genes in Bacillus subtilis and Pseudomonas putida. Fifteen open reading frames (ORFs) and 11 ORFs were identified in the 13.6 kb and the 9.8 kb fragments in B. subtilis and P. putida, respectively. Eight consecutive P. putida genes, except for one small ORF (homologous to gene 9K of Escherichia coli) in between, are homologous in sequence and relative locations to genes in B. subtilis. Altogether, 12 genes and their organization are conserved in B. subtilis and P. putida in the origin region. We found that the conserved region terminated on one side after the orf290 in P. putida (orf282 in B. subtilis). In the B. subtilis chromosome, five additional ORFs were found in between the conserved genes, suggesting that they are added after Gram‐positive bacteria were diverged from the Gram‐negative bacteria. One of the ORFs is a duplicate of the conserved gene. The third non‐translatable region containing multiple repeats of DnaA‐box (second in the case of P. putida) was found flanking gidA in both organisms. This result shows clearly that E. coli oriC and flanking genes gidA and gidB have been translocated by the inversion of some 40 kb fragment.
Cloning, sequencing and deletion from the chromosome of the gene encoding subunit I of the aa3‐type cytochrome c oxidase of Rhodobacter sphaeroides
Authors: Shapleigh James P. , Gennis Robert B.
Published: October 2006

The ctaD gene encoding subunit I of the aa3‐type cytochrome c oxidase from Rhodobacter sphaeroides has been cloned. The gene encodes a polypeptide of 565 residues which is highly homologous to the sequences of subunit I from other prokaryotic and eukaryotic sources, e.g. 51% identity with that from bovine, and 75% identity with that from Paracoccus denitrificans. The ctaD gene was deleted from the chromosome of R. sphaeroides, resulting in a strain that spectroscopically lacks cytochrome a. This strain maintains about 50% of the cytochrome c oxidase activity of the wild‐type strain owing to the presence of an alternate o‐type cytochrome c oxidase. The aa3‐type oxidase was restored by complementing the chromosomal deletion with a plasmid‐borne copy of the CtaD gene. This system is well suited for site‐directed mutagenesis probing of the structure and function of cytochrome c oxidase.