bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dissecting the loci of low‐level quinine resistance in malaria parasites
Authors: Ferdig Michael T. , Cooper Roland A. , Mu Jianbing , Deng Bingbing , Joy Deirdre A. , Su Xin‐zhuan , Wellems Thomas E. 
Published: May 2004

SummaryQuinine (QN) remains effective against Plasmodium falciparum, but its decreasing efficacy is documented from different continents. Multiple genes are likely to contribute to the evolution of QN resistance. To locate genes contributing to QN response variation, we have searched a P. falciparum genetic cross for quantitative trait loci (QTL). Results identify additive QTL in segments of chromosomes (Chrs) 13, 7 and 5, and pairwise effects from two additional loci of Chrs 9 and 6 that interact, respectively, with the QTL of Chrs 13 and 7. The mapped segments of Chrs 7 and 5 contain pfcrt, the determinant of chloroquine resistance (CQR), and pfmdr1, a gene known to affect QN responses. Association of pfcrt with a QTL of QN resistance supports anecdotal evidence for an evolutionary relationship between CQR and reduced QN sensitivity. The Chr 13 segment contains several candidate genes, one of which (pfnhe‐1) encodes a putative Na+/H+ exchanger. A repeat polymorphism in pfnhe‐1 shows significant association with low QN response in a collection of P. falciparum strains from Asia, Africa and Central and South America. Dissection of the genes and modifiers involved in QN response will require experimental strategies that can evaluate multiple genes from different chromosomes in combination.
Environmental signals and regulatory pathways that influence biofilm formation
Authors: Stanley Nicola R. , Lazazzera Beth A. 
Published: May 2004

SummaryIn nature, bacteria often exist as biofilms. Here, we discuss the environmental signals and regulatory proteins that affect both the initiation of bacterial biofilm formation and the nature of the mature biofilm structure. Current research suggests that the environmental signals regulating whether bacterial cells will initiate a biofilm differ from one bacterial species to another. This may allow each bacterial species to colonize its preferred environment efficiently. In contrast, some of the environmental signals that have currently been identified to regulate the structure of a mature biofilm are nutrient availability and quorum sensing, and are not species specific. These environmental signals evoke changes in the nature of the mature biofilm that may ensure optimal nutrient acquisition. Nutrient availability regulates the depth of the biofilm in such a way that the maximal number of cells in a biofilm appears to occur at suboptimal nutrient concentrations. At either extreme, nutrient‐rich or very nutrient‐poor conditions, greater numbers of cells are in the planktonic phase where they have greater access to the local nutrients or can be distributed to a new environment. Similarly, quorum‐sensing control of the formation of channels and pillar‐like structures may ensure efficient nutrient delivery to cells in a biofilm.
IS6110 functions as a mobile, monocyte‐activated promoter in Mycobacterium tuberculosis
Authors: Safi Hassan , Barnes Peter F. , Lakey David L. , Shams Homayoun , Samten Buka , Vankayalapati Ramakrishna , Howard Susan T. 
Published: May 2004

SummaryThe mobile insertion sequence, IS6110, is an important marker in tracking of Mycobacterium tuberculosis strains. Here, we demonstrate that IS6110 can upregulate downstream genes through an outward‐directed promoter in its 3′ end, thus adding to the significance of this element. Promoter activity was orientation dependent and was localized within a 110 bp fragment adjacent to the right terminal inverted repeat. Transcripts from this promoter, named OP6110, begin ≈ 85 bp upstream of the 3′ end of IS6110. Use of green fluorescent protein (GFP) expression constructs showed that OP6110 was upregulated in M. tuberculosis during growth in human monocytes and in late growth phases in broth. Analysis of natural insertion sites in M. tuberculosis showed that IS6110 upregulated expression of several downstream genes during growth in human monocytes, including Rv2280 in H37Rv and the PE‐PGRS gene, Rv1468c, in the clinical strain 210, which is a member of the Beijing family. Transcription between IS6110 and downstream genes was confirmed by reverse transcription polymerase chain reaction. The ability to activate genes during infection suggests that IS6110 has the potential to influence growth characteristics of different strains, and indicates another mechanism by which IS6110 can impact M. tuberculosis evolution.
Phage T4 early promoters are resistant to inhibition by the anti‐sigma factor AsiA
Authors: Orsini Gilbert , Igonet Sébastien , Pène Carole , Sclavi Bianca , Buckle Malcolm , Uzan Marc , Kolb Annie 
Published: May 2004

SummaryPhage T4 early promoters are transcribed in vivo and in vitro by the Escherichia coli RNA polymerase holoenzyme Eσ70. We studied in vitro the effects of the T4 anti‐σ70 factor AsiA on the activity of several T4 early promoters. In single‐round transcription, promoters motB, denV, mrh.2, motA wild type and UP element‐deleted motA are strongly resistant to inhibition by AsiA. The α‐C‐terminal domain of Eσ70 is crucial to this resistance. DNase I footprinting of Eσ70 and Eσ70AsiA on motA and mrh.2 shows extended contacts between the holoenzyme with or without AsiA and upstream regions of these promoters. A TG → TC mutation of the extended −10 motif in the motA UP element‐deleted promoter strongly increases susceptibility to inhibition by AsiA, but has no effect on the motA wild‐type promoter: either the UP element or the extended −10 site confers resistance to AsiA. Potassium permanganate reactivity shows that the two structure elements are not equivalent: with AsiA, the motA UP element‐deleted promoter opens more slowly whereas the motA TC promoter opens like the wild type. Changes in UV laser photoreactivity at position +4 on variants of motA reveal an analogous distinction in the roles of the extended −10 and UP promoter elements.
Transcriptional fidelity and proofreading in Archaea and implications for the mechanism of TFS‐induced RNA cleavage
Authors: Lange Udo , Hausner Winfried 
Published: May 2004

SummaryWe have addressed the question whether TFS, a protein that stimulates the intrinsic cleavage activity of the archaeal RNA polymerase, is able to improve the fidelity  of  transcription  in  Methanococcus.  Using non‐specific transcription experiments, we could demonstrate that misincorporation of non‐templated nucleotides  is  reduced  in  the  presence  of  TFS.  A more detailed analysis revealed that elongation complexes containing a misincorporated nucleotide were arrested, but could be reactivated by TFS. RNase as well as exonuclease III footprinting experiments demonstrated that this arrest was not combined with extended backtracking. Analysis of paused elongation complexes demonstrated that TFS is able to induce a cleavage resynthesis cycle in such complexes, which resulted in the accumulation of dinucleotides corresponding to the last two nucleotides of the transcript. Further analysis of cleavage products revealed that, even under conditions that strongly promote misincorporation, still 50% of the released dinucleotides were correctly incorporated. Therefore, we assume that pausing of elongation complexes is an important determinant of TFS‐induced RNA cleavage from the 3′ end. As the incorporation rate of wrong nucleotides is about 700‐fold reduced, it is possible that this delay also provides an appropriate time window for cleavage induction in order to maintain transcriptional fidelity by preventing misincorporation.
Proteomic screening and identification of differentially distributed membrane proteins in Escherichia coli
Authors: Lai Erh‐Min , Nair Usha , Phadke Nikhil D. , Maddock Janine R. 
Published: May 2004

SummaryBacteria show asymmetric subcellular distribution of many proteins involved in diverse cellular processes such as chemotaxis, motility, actin polymerization, chromosome partitioning and cell division. In many cases, the specific subcellular localization of these proteins is critical for proper regulation and function. Although cellular organization of the bacterial cell clearly plays an important role in cell physiology, systematic studies to uncover asymmetrically distributed proteins have not been reported previously. In this study, we undertook a proteomics approach to uncover polar membrane proteins in Escherichia coli. We identified membrane proteins enriched in E. coli minicells using a combination of two‐dimensional electrophoresis and mass spectrometry. Among a total of 173 membrane protein spots that were consistently detected, 36 spots were enriched in minicell membranes, whereas 15 spots were more abundant in rod cell membranes. The minicell‐enriched proteins included the inner membrane proteins MCPs, AtpA, AtpB, YiaF and AcrA, the membrane‐associated FtsZ protein and the outer membrane proteins YbhC, OmpW, Tsx, Pal, FadL, OmpT and BtuB. We immunolocalized two of the minicell‐enriched proteins, OmpW and YiaF, and showed that OmpW is a bona fide polar protein whereas YiaF displays a patchy membrane distribution with a polar and septal bias.
Molecular analysis of the key cytokinetic components of cyanobacteria: FtsZ, ZipN and MinCDE
Authors: Mazouni Khalil , Domain Francis , Cassier‐Chauvat Corinne , Chauvat Franck 
Published: May 2004

SummaryUsing a bacterial two‐hybrid system and a combination of in vivo and in vitro assays that take advantage of the green fluorescent reporter protein (GFP), we have investigated the localization and the protein–protein interaction of several key components of the cytokinetic machinery of cyanobacteria (i.e. the progenitor of chloroplast). We demonstrate that (i) the ftsZ and zipN genes are essential for the viability of the model cyanobacterium Synechocystis sp. PCC 6803, whereas the minCDE cluster is dispensable for cell growth; (ii) the GTP‐binding domain of FtsZ is crucial to FtsZ assembly into the septal ring at mid‐cell; (iii) the Z‐ring of deeply constricted daughter cells is oriented perpendicularly to the mother Z‐ring, showing that Synechocystis divides in alternating perpendicular planes; (iv) the MinCDE system affects the morphology of the cell, as well as the position and the shape of FtsZ structures; and (v) MinD is targeted to cell membranes in a process involving its C‐terminal amphipathic helix, but not its ATP‐binding region. Finally, we have also characterized a novel Z‐interacting protein, ZipN, the N‐terminal DnaJ domain of which is critical to the decoration of the Z‐ring, and we report that this process is independent of MinCDE.
An amino‐terminal domain of Enterococcus faecalis aggregation substance is required for aggregation, bacterial internalization by epithelial cells and binding to lipoteichoic acid
Authors: Waters Christopher M. , Hirt Helmut , McCormick John K. , Schlievert Patrick M. , Wells Carol L. , Dunny G. M. 
Published: May 2004

SummaryAggregation substance (AS), a plasmid‐encoded surface protein of Enterococcus faecalis, plays important roles in virulence and antibiotic resistance transfer. Previous studies have suggested that AS‐mediated aggregation of enterococcal cells could involve the binding of this protein to cell wall lipoteichoic acid (LTA). Here, a method to purify an undegraded form of Asc10, the AS of the plasmid pCF10, is described. Using this purified protein, direct binding of Asc10 to purified E. faecalis LTA was demonstrated. Equivalent binding of Asc10 to LTA purified from INY3000, an E. faecalis strain that is incapable of aggregation, was also observed. Surprisingly, mutations in a previously identified aggregation domain from amino acids 473 to 683 that abolished aggregation had no effect on LTA binding. In frame deletion analysis of Asc10 was used to identify a second aggregation domain located in the N‐terminus of the protein from amino acids 156 to 358. A purified Asc10 mutant protein lacking this domain showed reduced LTA binding, while a purified N‐terminal fragment from amino acids 44–331 had high LTA binding. Like the previously described aggregation domain, the newly identified Asc10(156−358) aggregation domain was also required for efficient internalization of E. faecalis into HT‐29 enterocytes. Thus, Asc10 possess two distinct domains required for aggregation and eukaryotic cell internalization: an N‐terminal domain that promotes binding to LTA and a second domain located near the middle of the protein.
Enhancement and rescue of target capture in Tn10 transposition by site‐specific modifications in target DNA
Authors: Pribil Patrick A. , Wardle Simon J. , Haniford David B. 
Published: May 2004

SummaryThe bacterial transposon Tn10 inserts preferentially into specific target sequences. This insertion specificity appears to be linked to the ability of target sites to adopt symmetrically positioned DNA bends after binding the transposition machinery. Target DNA bending is thought to permit the transposase protein to make additional contacts with the target DNA, thereby stabilizing the target complex so that the joining of transposon and target DNA sequences can occur efficiently. In the current work, we have asked whether the introduction of a discontinuity in a target DNA strand, a modification that is expected to make it easier for a DNA molecule to bend, can enhance or rescue target capture under otherwise suboptimal reaction conditions. We show that either a nick or a missing phosphate specifically at the site of reaction chemistry increases the ability of various target DNAs to form the target capture complex. The result suggests that the bends in the target DNA are highly localized and include the scissile phosphates. This raises the possibility that strand transfer is mechanistically linked to target capture. We have also identified specific residues in the target DNA and in transposase that appear to play an important role in target DNA bending.
Untangling intracellular DNA topology
Authors: Espeli Olivier , Marians Kenneth J. 
Published: May 2004

SummaryThe biochemical steps by which bacterial topoisomerases alter the topology of DNA are well known. However, it has been a more vexing task to establish physiological roles and sites of action of the different topoisomerases within the context of the bacterial cell cycle. This difficulty can be attributed in part to the redundancy among the activities of the different enzymes. In this microreview, we will focus on recent progress in understanding the topological structure of the chromosome, analysis of topoisomerase mechanism in single‐molecule assays and recent data on the regulation and integration of topoisomerase activity within the cell cycle that have all brought a new perspective to the action of topoisomerases in the bacterial cell.
Quantitative proteomics of the human malaria parasite Plasmodium falciparum and its application to studies of development and inhibition
Authors: Nirmalan Niroshini , Sims Paul F. G. , Hyde John E. 
Published: May 2004

SummaryThe ability to measure accurately comparative levels of protein expression after drug challenge, metabolic stress, developmental programming or other perturbation represents one of the most important goals in post‐genomics malaria research. We describe here a simple and robust quantitative methodology that is ideally suited to in vitro experiments designed to study changes in the proteome of the most important of the human parasites, the lethal species Plasmodium falciparum. The metabolic labelling technique we have developed uses parasite uptake of heavy isotope‐containing isoleucine during normal growth followed by two‐dimensional separation of individual proteins and mass spectrometry. The method is applicable to essentially each of the ≈ 5300 proteins of P. falciparum predicted from the completed genome sequence, permitting facile identification and accurate comparative quantification of labelled peptides from any of these proteins synthesized by in vitro cultures subjected to different stimuli. We demonstrate its application to the study of cell cycle changes, where we observe divergent patterns of protein and reported transcript levels indicative of modulation at the translational level. Our data also provide evidence for significant levels of post‐translational modification in the parasite, and we measure differences among variants of phosphoethanolamine N‐methyltransferase and actin‐I across the cell cycle. We have also monitored parasite responses to equipotent doses of the clinical antimalarial inhibitors pyrimethamine and tetracycline and observed differential effects for a number of proteins unrelated to likely targets of these drugs.
Branching sites and morphological abnormalities behave as ectopic poles in shape‐defective Escherichia coli
Authors: Nilsen Trine , Ghosh Anindya S. , Goldberg Marcia B. , Young Kevin D. 
Published: May 2004

SummaryCertain mutants in Escherichia coli lacking multiple penicillin‐binding proteins (PBPs) produce misshapen cells containing kinks, bends and branches. These deformed regions exhibit two structural characteristics of normal cell poles: the peptidoglycan is inert to dilution by new synthesis or turnover, and a similarly stable patch of outer membrane caps the sites. To test the premise that these aberrant sites represent biochemically functional but misplaced cell poles, we assessed the intracellular distribution of proteins that localize specifically to bacterial poles. Green fluorescent protein (GFP) hybrids containing polar localization sequences from the Shigella flexneri IcsA protein or from the Vibrio cholerae EpsM protein formed foci at the poles of wild‐type E. coli and at the poles and morphological abnormalities in PBP mutants. In addition, secreted wild‐type IcsA localized to the outer membrane overlying these aberrant domains. We conclude that the morphologically deformed sites in these mutants represent fully functional poles or pole fragments. The results suggest that prokaryotic morphology is driven, at least in part, by the controlled placement of polar material, and that one or more of the low‐molecular‐weight PBPs participate in this process. Such mutants may help to unravel how particular proteins are targeted to bacterial poles, thereby creating important biochemical and functional asymmetries.
The effect of host‐encoded nucleoid proteins on transposition: H‐NS influences targeting of both IS903 and Tn10
Authors: Swingle Bryan , O'Carroll Michelle , Haniford David , Derbyshire Keith M. 
Published: May 2004

SummaryNucleoid proteins are small, abundant, DNA‐binding proteins that profoundly affect the local and global structure of the chromosome, and play a major role in gene regulation. Although several of these proteins have been shown to enhance assembly of transpososomes before initiating transposition, no systematic survey has been carried out examining the in vivo role(s) of these proteins in transposition. We have examined the requirement of the six most abundant nucleoid proteins in transposition for three different transposons, IS903, Tn10 and Tn552. Most notably, H‐NS was required for efficient transposition of all three elements in a papillation assay, suggesting a general role for H‐NS in bacterial transposition. Further studies indicated that H‐NS was exerting its effect on target capture. Targeting preferences for IS903 into the Escherichia coli chromosome were dramatically altered in the absence of H‐NS. In addition, the alterations observed in the IS903 target profile emphasized the important role that H‐NS plays in chromosome organization. A defect in target capture was also inferred for Tn10, as an excised transposon fragment, a precursor to target capture, accumulated in in vivo induction assays. Furthermore, a transposase mutant that is known to increase target DNA bending and to relax target specificity eliminated this block to target capture. Together, these results imply a role for H‐NS in target capture, either by providing regions of DNA more accessible to transposition or by stabilizing transpososome binding to captured targets immediately before strand transfer.
Regulation of type III secretion in Bordetella
Authors: Mattoo Seema , Yuk Ming H. , Huang Lisa L. , Miller Jeff F. 
Published: May 2004

SummaryThe BvgAS virulence control system regulates the expression of type III secretion genes in Bordetella subspecies that infect humans and other mammals. We have identified five open reading frames, btrS, btrU, btrX, btrW and btrV, that are activated by BvgAS and encode regulatory factors that control type III secretion at the levels of transcription, protein expression and secretion. The btrS gene product bears sequence similarity to ECF (extracytoplasmic function) sigma factors and is required for transcription of the bsc locus. btrU, btrW and btrV encode proteins predicted to contain PP2C‐like Ser phosphatase, HPK (His protein kinase)‐like Ser kinase and STAS anti‐sigma factor antagonist domains, respectively, which are characteristic of Gram‐positive partner switching proteins in Bacillus subtilis. BtrU and BtrW are required for secretion of proteins that are exported by the bsc type III secretion system, whereas BtrV is specifically required for protein synthesis and/or stability. Bordetella species have thus evolved a unique cascade to differentially regulate type III secretion that combines a canonical phosphorelay system with an ECF sigma factor and a set of proteins with domain signatures that define partner switchers, which were traditionally thought to function only in Gram‐positive bacteria. The presence of multiple layers and mechanisms of regulation most likely reflects the need to integrate multiple signals in controlling type III secretion. The bsc and btr loci are nearly identical between broad‐host‐range and human‐specific Bordetella. Comparative analysis of Bordetella subspecies revealed that, whereas bsc and btr loci were transcribed in all subspecies, only broad‐host‐range strains expressed a functional type III secretion system in vitro. The block in type III secretion is post‐transcriptional in human‐adapted strains, and signal recognition appears to be a point of divergence between subspecies.
Cell‐to‐cell signalling in Escherichia coli and Salmonella enterica
Authors: Ahmer Brian M. M. 
Published: May 2004

SummaryCell‐to‐cell signalling in prokaryotes that leads to co‐ordinated behaviour has been termed quorum sensing. This type of signalling can have profound impacts on microbial community structure and host–microbe interactions. The Gram‐negative quorum‐sensing systems were first discovered and extensively characterized in the marine Vibrios. Some components of the Vibrio systems are present in the classical genetic model organisms Escherichia coli and Salmonella enterica. Both organisms encode a signal receptor of the LuxR family, SdiA, but not a corresponding signal‐generating enzyme. Instead, SdiA of Salmonella detects and responds to signals generated only by other microbial species. Conversely, E. coli and Salmonella encode the signal‐generating component of a second system (a LuxS homologue that generates AI‐2), but the sensory apparatus for AI‐2 differs substantially from the Vibrio system. The only genes currently known to be regulated by AI‐2 in Salmonella encode an active uptake and modification system for AI‐2. Therefore, it is not yet clear whether Salmonella uses AI‐2 as a signal molecule or whether AI‐2 has some other function. In E. coli, the functions of both SdiA and AI‐2 are unclear due to pleiotropy. Genetic strategies to identify novel signalling systems have been performed with E. coli and Providencia stuartii. Several putative signalling systems have been identified, one that uses indole as a signal and another that releases what appears to be a peptide. The latter system has homologues in E. coli and Salmonella, as well as other bacteria, plants and animals. In fact, the protease components from Providencia and Drosophila are functionally interchangeable.