bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular and biochemical characterization of a calcium/calmodulin-binding protein kinase from rice
Authors: ZHANG Lei , LIU Bi-Feng , LIANG Shuping , JONES Russell L. , LU Ying-Tang
Published: November 2002

A Ca2+/calmodulin (CaM)-binding protein kinase from rice (Oryza sativa), OsCBK, has been characterized that lacks Ca2+-binding EF hands and has Ca2+/CaM-independent autophosphorylation and substrate-phosphorylation activity. OsCBK has all 11 subdomains of a kinase catalytic domain and a putative CaM-binding domain, and shares high identity with Ca2+-dependent-protein-kinase ('CDPK')-related protein kinases in plants. OsCBK bound CaM in a Ca2+-dependent manner as previously reported for Ca2+/calmodulin-dependent protein kinases in animals, but autophosphorylation and phosphorylation of histone IIIs were Ca2+/CaM-independent. Surface plasmon resonance analysis showed that OsCBK specifically bound CaM with high affinity (KD = 30nM). Capillary electrophoresis showed that phosphorylation of OsCBK occurred on serine and threonine residues. These data show that OsCBK is a serine/threonine protein kinase that binds Ca2+/CaM, but whose enzymic activity is independent of Ca2+/CaM. In situ hybridization showed that OsCBK is expressed in reproductive and vegetative tissues of rice and shows temporal and spatial changes during plant growth and development. OsCBK is highly expressed in zones of cell division and it is particularly abundant in sporogenous cells of the anther at meiosis.
Identification of in vitro and in vivo phosphorylation sites in the catalytic subunit of the DNA-dependent protein kinase
Authors: DOUGLAS Pauline , SAPKOTA Gopal P. , MORRICE Nick , YU Yaping , GOODARZI Aaron A. , MERKLE Dennis , MEEK Katheryn , ALESSI Dario R. , LEES-MILLER Susan P.
Published: November 2002

The DNA-dependent protein kinase (DNA-PK) is required for the repair of DNA double-strand breaks (DSBs), such as those caused by ionizing radiation and other DNA-damaging agents. DNA-PK is composed of a large catalytic subunit (DNA-PKcs) and a heterodimer of Ku70 and Ku80 that assemble on the ends of double-stranded DNA to form an active serine/threonine protein kinase complex. Despite in vitro and in vivo evidence to support an essential role for the protein kinase activity of DNA-PK in the repair of DNA DSBs, the physiological targets of DNA-PK have remained elusive. We have previously shown that DNA-PK undergoes autophosphorylation in vitro, and that autophosphorylation correlates with loss of protein kinase activity and dissociation of the DNA-PK complex. Also, treatment of cells with the protein phosphatase inhibitor, okadaic acid, enhances DNA-PKcs phosphorylation and reduces DNA-PK activity in vivo. Here, using solid-phase protein sequencing, MS and phosphospecific antibodies, we have identified seven in vitro autophosphorylation sites in DNA-PKcs. Six of these sites (Thr2609, Ser2612, Thr2620, Ser2624, Thr2638 and Thr2647) are clustered in a region of 38 amino acids in the central region of the protein. Five of these sites (Thr2609, Ser2612, Thr2638, Thr2647 and Ser3205) are conserved between six vertebrate species. Moreover, we show that DNA-PKcs is phosphorylated in vivo at Thr2609, Ser2612, Thr2638 and Thr2647 in okadaic acid-treated human cells. We propose that phosphorylation of these sites may play an important role in DNA-PK function.
Far-red fluorescent tag for protein labelling
Authors: FRADKOV Arkady F. , VERKHUSHA Vladislav V. , STAROVEROV Dmitry B. , BULINA Maria E. , YANUSHEVICH Yurii G. , MARTYNOV Vladimir I. , LUKYANOV Sergey , LUKYANOV Konstantin A.
Published: November 2002

Practical applications of green fluorescent protein ('GFP')-like fluorescent proteins (FPs) from species of the class Anthozoa (sea anemones, corals and sea pens) are strongly restricted owing to their oligomeric nature. Here we suggest a strategy to overcome this problem by the use of two covalently linked identical red FPs as non-oligomerizing fusion tags. We have applied this approach to the dimeric far-red fluorescent protein HcRed1 and have demonstrated superiority of the tandem tag in the in vivo labelling of fine cytoskeletal structures and tiny nucleoli. In addition, a possibility of effective fluorescence resonance energy transfer ('FRET') between enhanced yellow FP mutant ('EYFP') and tandem HcRed1 was demonstrated in a protease assay.
Topological quantum computation
Authors: Freedman Michael , Kitaev Alexei , Larsen Michael , Wang Zhenghan
Published: November 2002

The theory of quantum computation can be constructed from the abstract study of anyonic systems. In mathematical terms, these are unitary topological modular functors. They underlie the Jones polynomial and arise in Witten-Chern-Simons theory. The braiding and fusion of anyonic excitations in quantum Hall electron liquids and 2D-magnets are modeled by modular functors, opening a new possibility for the realization of quantum computers. The chief advantage of anyonic computation would be physical error correction: An error rate scaling likee−αℓe^{-\alpha \ell }, whereℓ\ellis a length scale, andα\alphais some positive constant. In contrast, the “presumptive" qubit-model of quantum computation, which repairs errors combinatorically, requires a fantastically low initial error rate (about10−410^{-4}) before computation can be stabilized.
Physical law and the quest for mathematical understanding
Authors: Witten Edward
Published: November 2002

The theoretical physics of the first quarter of the twentieth century —centering around relativity theory and nonrelativistic quantum mechanics—has had a broad influence mathematically. The main achievement of theoretical physics in the following half-century was the development of quantum field theory or QFT. Yet the mathematical influence of QFT still belongs largely to the

21

s
t

21^{st}

century, because its mathematical foundations are still not well-understood.
MethPrimer: designing primers for methylation PCRs
Authors: Li Long-Cheng , Dahiya Rajvir
Published: November 2002

Motivation: DNA methylation is an epigenetic mechanism of gene regulation. Bisulfite- conversion-based PCR methods, such as bisulfite sequencing PCR (BSP) and methylation specific PCR (MSP), remain the most commonly used techniques for methylation mapping. Existing primer design programs developed for standard PCR cannot handle primer design for bisulfite-conversion-based PCRs due to changes in DNA sequence context caused by bisulfite treatment and many special constraints both on the primers and the region to be amplified for such experiments. Therefore, the present study was designed to develop a program for such applications.

Results: MethPrimer, based on Primer3, is a program for designing PCR primers for methylation mapping. It first takes a DNA sequence as its input and searches the sequence for potential CpG islands. Primers are then picked around the predicted CpG islands or around regions specified by users. MethPrimer can design primers for BSP and MSP. Results of primer selection are delivered through a web browser in text and in graphic view.

Availability: MethPrimer is freely accessible at the following Web address http://itsa.ucsf.edu/~urolab/methprimer

Contact: longli@itsa.ucsf.eduurologylab@aol.com

* To whom correspondence should be addressed.
Nonparametric methods for identifying differentially
expressed genes in microarray data
Authors: Troyanskaya Olga G. , Garber Mitchell E. , Brown Patrick O. , Botstein David , Altman Russ B.
Published: November 2002

Motivation: Gene expression experiments provide a fast and systematic way to identify disease markers relevant to clinical care. In this study, we address the problem of robust identification of differentially expressed genes from microarray data. Differentially expressed genes, or discriminator genes, are genes with significantly different expression in two user-defined groups of microarray experiments. We compare three model-free approaches: (1) nonparametric t-test, (2) Wilcoxon (or Mann–Whitney) rank sum test, and (3) a heuristic method based on high Pearson correlation to a perfectly differentiating gene (‘ideal discriminator method’). We systematically assess the performance of each method based on simulated and biological data under varying noise levels and p-value cutoffs.

Results: All methods exhibit very low false positive rates and identify a large fraction of the differentially expressed genes in simulated data sets with noise level similar to that of actual data. Overall, the rank sum test appears most conservative, which may be advantageous when the computationally identified genes need to be tested biologically. However, if a more inclusive list of markers is desired, a higher p-value cutoff or the nonparametric t-test may be appropriate. When applied to data from lung tumor and lymphoma data sets, the methods identify biologically relevant differentially expressed genes that allow clear separation of groups in question. Thus the methods described and evaluated here provide a convenient and robust way to identify differentially expressed genes for further biological and clinical analysis.

Availability: By request from the authors.

Contact: russ.altman@stanford.edu

* To whom correspondence should be addressed.
An evaluation of β-turn prediction methods
Authors: Kaur Harpreet , Raghava G.P.S.
Published: November 2002

Motivation: β-turn is an important element of protein structure. In the past three decades, numerous β-turn prediction methods have been developed based on various strategies. For a detailed discussion about the importance of β-turns and a systematic introduction of the existing prediction algorithms for β-turns and their types, please see a recent review (Chou, Analytical Biochemistry, 286, 1–16, 2000). However at present, it is still difficult to say which method is better than the other. This is because of the fact that these methods were developed on different sets of data. Thus, it is important to evaluate the performance of β-turn prediction methods.

Results: We have evaluated the performance of six methods of β-turn prediction. All the methods have been tested on a set of 426 non-homologous protein chains. It has been observed that the performance of the neural network based method, BTPRED, is significantly better than the statistical methods. One of the reasons for its better performance is that it utilizes the predicted secondary structure information. We have also trained, tested and evaluated the performance of all methods except BTPRED and GORBTURN, on new data set using a 7-fold cross-validation technique. There is a significant improvement in performance of all the methods when secondary structure information is incorporated. Moreover, after incorporating secondary structure information, the Sequence Coupled Model has yielded better results in predicting β-turns as compared with other methods. In this study, both threshold dependent and independent (ROC) measures have been used for evaluation.

Contact: raghava@imtech.res.in

Supplementary information: http://imtech.res.in/raghava/betatpred/eval/

* To whom correspondence should be addressed.
Euclidian space and grouping of biological objects
Authors: Grishin Vyacheslav N. , Grishin Nick V.
Published: November 2002

Motivation: Biological objects tend to cluster into discrete groups. Objects within a group typically possess similar properties. It is important to have fast and efficient tools for grouping objects that result in biologically meaningful clusters. Protein sequences reflect biological diversity and offer an extraordinary variety of objects for polishing clustering strategies. Grouping of sequences should reflect their evolutionary history and their functional properties. Visualization of relationships between sequences is of no less importance. Tree-building methods are typically used for such visualization. An alternative concept to visualization is a multidimensional sequence space. In this space, proteins are defined as points and distances between the points reflect the relationships between the proteins. Such a space can also be a basis for model-based clustering strategies that typically produce results correlating better with biological properties of proteins.

Results: We developed an approach to classification of biological objects that combines evolutionary measures of their similarity with a model-based clustering procedure. We apply the methodology to amino acid sequences. On the first step, given a multiple sequence alignment, we estimate evolutionary distances between proteins measured in expected numbers of amino acid substitutions per site. These distances are additive and are suitable for evolutionary tree reconstruction. On the second step, we find the best fit approximation of the evolutionary distances by Euclidian distances and thus represent each protein by a point in a multidimensional space. The Euclidian space may be projected in two or three dimensions and the projections can be used to visualize relationships between proteins. On the third step, we find a non-parametric estimate of the probability density of the points and cluster the points that belong to the same local maximum of this density in a group. The number of groups is controlled by a σ-parameter that determines the shape of the density estimate and the number of maxima in it. The grouping procedure outperforms commonly used methods such as UPGMA and single linkage clustering.

Availability: The code of EESG program for Mathematica4 (Wolfram Research) as well as the details of the analysis are freely available at ftp://iole.swmed.edu/pub/EESG/.

Contact: grishin@chop.swmed.edu

* To whom correspondence should be addressed.
A motif of a microbial starch-binding domain found in human genethonin
Authors: Janeček Štefan
Published: November 2002

Summary: The sequence of the starch-binding domain present in 10% of amylolytic enzymes of microbial origin and classified as the carbohydrate-binding module family 20, was identified in the equivalent part of sequence of human genethonin, a skeletal muscle protein of unknown function. The sequence identity between the starch-binding domain from Bacillus sp. strain 1011 cyclodextrin glucanotransferase and the corresponding segment of human genethonin was higher than 28%. The amino acid residues known to be involved in the raw starch binding were found to be conserved in the genethonin sequence. The three-dimensional structure of the genethonin ‘starch-binding domain’ was modelled and its eventual function briefly discussed.

Contact: stefan.janecek@savba.sk
Poxvirus Orthologous Clusters (POCs)
Authors: Ehlers Angelika , Osborne John , Slack Stephanie , Roper Rachel L. , Upton Chris
Published: November 2002

Summary: Poxvirus Orthologous Clusters (POCs) is a JAVA client–server application which accesses an updated database containing all complete poxvirus genomes; it automatically groups orthologous genes into families based on BLASTP scores for assessment by a human database curator. POCs has a user-friendly interface permitting complex SQL queries to retrieve interesting groups of DNA and protein sequences as well as gene families for subsequent interrogation by a variety of integrated tools: BLASTP, BLASTX, TBLASTN, Jalview (multiple alignment), Dotlet (Dotplot), Laj (local alignment), and NAP (nucleotide to amino acid alignment).

Availability: Direct access to the POCs database via the GENOME ANALYSIS link at the Poxvirus Bioinformatics Resource: http://www.poxvirus.org. The Software is available for download via HTTP at: http://athena.bioc.uvic.ca/pbr/POCs/pocs.html.

Contact: cupton@uvic.ca

Supplementary Information: Installation instructions, the User's Manual, screenshots, and examples are available at the POCs home page http://athena.bioc.uvic.ca/pbr/POCs/pocs.html. The software is free for non-commercial applications. For information on poxviruses see http://www.poxvirus.org.

* To whom correspondence should be addressed.
Unexpected molecular structure from laboratory powder diffraction data
Authors: Chernyshev Vladimir V. , Yatsenko Alexandr V. , Kuvshinov Alexandr M. , Shevelev Svyatoslav A.
Published: November 2002

In the framework of a thorough study of the reactions ofC-(2,4,6-trinitrophenyl)azomethines with various nucleophiles, the crystal and molecular structures of three compounds, namely 2,4-dinitro-N-phenyl-6-(phenylazo)benzamide, (I),N-(2-methoxyphenyl)-2-(2-methoxyphenylazo)-4,6-dinitrobenzamide, (II), andN-methyl-2,4-dinitro-N-phenyl-6-(phenylazo)benzamide, (III), were determined from low-resolution laboratory powder diffraction data. The crystal structure determination of (I), starting from erroneous two-dimensional structures, produced the correct solution as a result of the use of a combination of a systematic grid search and bond-restrained Rietveld refinement. The obtained molecular structure of (I) was quite unexpected. Based on the correct molecular structure of (I), the crystal structures of (II) and (III) were routinely solved by the grid search technique.
An acceptance-diagram description of beams from focusing monochromators
Authors: Cussen L. D.
Published: November 2002

`Acceptance' diagrams are used to describe the beams produced by curved segmented (`focusing') monochromators in the kinematic approximation. This semi-analytic semi-graphical method leads to a complete description which is readily visualized in terms of instrument variables. Focusing both in and perpendicular to the scattering plane is considered. Clear relationships are identified between instrument dimensions and the beam produced. The description is directed towards neutron scattering instruments but the formalism should also apply to photons and electrons. It is demonstrated that a monochromator curved both in and perpendicular to the scattering plane (`double focused') on either a straight or curved neutron guide tube from a reactor source can produce a beam of comparable flux to that achievable at the reactor face. Such a beam has spatial dimensions comparable with modern single-crystal samples for inelastic scattering and could thus provide spectacular signal and, even more interestingly, signal to noise ratios for both elastic and inelastic single-crystal spectrometers.
Online versus offline research: implications for evaluating digital media
Authors: Gunter Barrie , Nicholas David , Huntington Paul , Williams Peter
Published: November 2002

The growth of the Internet and other digital media has opened up exciting opportunities for the provision of public services, for business and for personal transactions. Comparisons between the earliest forms of “online” research, in the form of telephone interviewing, and offline data collection via face‐to‐face interviews or self‐completion questionnaires, revealed that the modality within which research was conducted could affect research findings. In examining the evidence, this paper indicates that the use of online methodologies has important implications for sampling, response rates, quality of data produced, and operational practices in research projects. Online research is restricted to individuals with access to relevant technologies (e.g. the Internet) and where online technology penetration is limited, survey samples are unlikely to represent the general population. Online surveys, however, can produce quicker response rates than offline surveys and also richer open‐ended responses. The important point is to recognise the strengths and weaknesses are associated with different methodologies and what differences can exist between online and offline data collection procedures.
The use of participatory research as an alternative approach for information needs research
Authors: Penzhorn Cecilia
Published: November 2002

This article reports on a study undertaken to explore the feasibility of using a participatory research approach as a method for determining information needs. Dissatisfaction with the use of quantitative methods and techniques in information needs research resulted in a gradual move towards the use of qualitative methods focusing on the users themselves in determining their information needs. Participatory research is a qualitative research methodology that challenges the principles and practices of objective, detached, quantitative research approaches. The use of participatory research methods in this project resulted in the identification of reliable and relevant information needs. These results can serve as an example for the increasing use of qualitative techniques in information needs research and affirm that participatory research methods can be a valuable alternative in the area of information needs research.
Restructuring library organizations for the twenty‐first century: the future of user‐oriented services in Korean academic libraries
Authors: Shin Eun‐Ja , Kim Young‐Seok
Published: November 2002

This article examines why Korean academic libraries embarked on restructuring their organization and identifies possible solutions to some problems that these libraries face. The conclusion is drawn from the results of a case study on Sejong University Library that Korean academic librarians should become user‐centered professionals. After restructuring, the user service librarian’s job content has changed to concentrate on servicing users on specific subjects. The subject librarian will take care of reference services, SDI, DB instruction and book selection on the same subject.
The IκB-NF-κB Signaling Module: Temporal Control and Selective Gene Activation
Authors: Hoffmann Alexander , Levchenko Andre , Scott Martin L. , Baltimore David
Published: November 2002

Nuclear localization of the transcriptional activator NF-κB (nuclear factor κB) is controlled in mammalian cells by three isoforms of NF-κB inhibitor protein: IκBα, -β, and -
ɛ
. Based on simplifying reductions of the IκB–NF-κB signaling module in knockout cell lines, we present a computational model that describes the temporal control of NF-κB activation by the coordinated degradation and synthesis of IκB proteins. The model demonstrates that IκBα is responsible for strong negative feedback that allows for a fast turn-off of the NF-κB response, whereas IκBβ and -
ɛ
function to reduce the system's oscillatory potential and stabilize NF-κB responses during longer stimulations. Bimodal signal-processing characteristics with respect to stimulus duration are revealed by the model and are shown to generate specificity in gene expression.
Macromolecular Architecture in Eukaryotic Cells Visualized by Cryoelectron Tomography
Authors: Medalia Ohad , Weber Igor , Frangakis Achilleas S. , Nicastro Daniela , Gerisch Günther , Baumeister Wolfgang
Published: November 2002

Electron tomography of vitrified cells is a noninvasive three-dimensional imaging technique that opens up new vistas for exploring the supramolecular organization of the cytoplasm. We applied this technique to
Dictyostelium
cells, focusing on the actin cytoskeleton. In actin networks reconstructed without prior removal of membranes or extraction of soluble proteins, the cross-linking of individual microfilaments, their branching angles, and membrane attachment sites can be analyzed. At a resolution of 5 to 6 nanometers, single macromolecules with distinct shapes, such as the 26
S
proteasome, can be identified in an unperturbed cellular environment.
Sperm-Female Coevolution in
Drosophila
Authors: Miller Gary T. , Pitnick Scott
Published: November 2002

Rapid evolution of reproductive traits has been attributed to sexual selection arising from interaction between the sexes. However, little is known about the nature of selection driving the evolution of interacting sex-specific phenotypes. Using populations of
Drosophila melanogaster
selected for divergent sperm length or female sperm-storage organ length, we experimentally show that male fertilization success is determined by an interaction between sperm and female morphology. In addition, sperm length evolution occurred as a correlated response to selection on the female reproductive tract. Giant sperm tails are the cellular equivalent of the peacock's tail, having evolved because females evolved reproductive tracts that selectively bias paternity in favor of males with longer sperm.
Seismic Images of Crust and Upper Mantle Beneath Tibet: Evidence for Eurasian Plate Subduction
Authors: Kind R. , Yuan X. , Saul J. , Nelson D. , Sobolev S. V. , Mechie J. , Zhao W. , Kosarev G. , Ni J. , Achauer U. , Jiang M.
Published: November 2002

Seismic data from central Tibet have been combined to image the subsurface structure and understand the evolution of the collision of India and Eurasia. The 410- and 660-kilometer mantle discontinuities are sharply defined, implying a lack of a subducting slab beneath the plateau. The discontinuities appear slightly deeper beneath northern Tibet, implying that the average temperature of the mantle above the transition zone is about 300°C hotter in the north than in the south. There is a prominent south-dipping converter in the uppermost mantle beneath northern Tibet that might represent the top of the Eurasian mantle lithosphere underthrusting the northern margin of the plateau.
Biochemical Properties of
Neisseria gonorrhoeae
LgtE
Authors: Piekarowicz Andrzej , Stein Daniel C.
Published: November 2002

A fragment of chromosomal DNA encoding the
lgtE
gene of
Neisseria gonorrhoeae
strain F62 was amplified by PCR and cloned into the expression vector pET15b. Functional LgtE was purified and its biochemical properties were determined. The purified enzyme was maximally active in buffer containing manganese; minimal activity was obtained in buffer containing other divalent cations. LgtE was only able to mediate the addition of UDP-galactose into neisserial lipooligosaccharides (LOSs). We used a variety of genetically defined and chemically verified LOS structures to determine acceptor specificity. LgtE was able to mediate the addition of galactose into a variety of LOS structures, indicating the this enzyme possesses broad acceptor specificity. Furthermore, it was able to add multiple galactose residues onto LOS. We also determined that this enzyme was capable of adding galactose onto both the α and β chains of neisserial LOS.
Requirement offlhAfor Swarming Differentiation, Flagellin Export, and Secretion of Virulence-Associated Proteins inBacillus thuringiensis
Authors: Ghelardi Emilia , Celandroni Francesco , Salvetti Sara , Beecher Douglas J. , Gominet Myriam , Lereclus Didier , Wong Amy C. L. , Senesi Sonia
Published: November 2002

Bacillus thuringiensisis being used worldwide as a biopesticide, although increasing evidence suggests that it is emerging as an opportunistic human pathogen. While phospholipases, hemolysins, and enterotoxins are claimed to be responsible forB. thuringiensisvirulence, there is no direct evidence to indicate that the flagellum-driven motility plays a role in parasite-host interactions. This report describes the characterization of a mini-Tn10mutant ofB. thuringiensisthat is defective in flagellum filament assembly and in swimming and swarming motility as well as in the production of hemolysin BL and phosphatidylcholine-preferring phospholipase C. The mutant strain was determined to carry the transposon insertion inflhA, a flagellar class II gene encoding a protein of the flagellar type III export apparatus. Interestingly, theflhAmutant ofB. thuringiensissynthesized flagellin but was impaired in flagellin export. Moreover, a protein similar to the anti-sigma factor FlgM that acts in regulating flagellar class III gene transcription was not detectable inB. thuringiensis, thus suggesting that the flagellar gene expression hierarchy ofB. thuringiensisdiffers from that described forBacillus subtilis. TheflhAmutant ofB. thuringiensiswas also defective in the secretion of hemolysin BL and phosphatidylcholine-preferring phospholipase C, although both of these virulence factors were synthesized by the mutant. Since complementation of the mutant with a plasmid harboring theflhAgene restored swimming and swarming motility as well as secretion of toxins, the overall results indicate that motility and virulence inB. thuringiensismay be coordinately regulated byflhA, which appears to play a crucial role in the export of flagellar as well as nonflagellar proteins.
Functions Required for Extracellular Quinolone Signaling by
Pseudomonas aeruginosa
Authors: Gallagher Larry A. , McKnight Susan L. , Kuznetsova Marina S. , Pesci Everett C. , Manoil Colin
Published: November 2002

A set of 30 mutants exhibiting reduced production of the phenazine poison pyocyanin were isolated following transposon mutagenesis of
Pseudomonas aeruginosa
PAO1. The mutants could be subdivided into those with defects in the primary phenazine biosynthetic pathway and those with more pleiotropic defects. The largest set of pleiotropic mutations blocked the production of the extracellular
Pseudomonas
quinolone signal (PQS), a molecule required for the synthesis of secondary metabolites and extracellular enzymes. Most of these
pqs
mutations affected genes which appear to encode PQS biosynthetic functions, although a transcriptional regulator and an apparent response effector were also represented. Two of the genes required for PQS synthesis (
phnA
and
phnB
) had previously been assumed to encode phenazine biosynthetic functions. The transcription of one of the genes required for PQS synthesis (PA2587/
pqsH
) was regulated by the LasI/R quorum-sensing system, thereby linking quorum sensing and PQS regulation. Others of the pleiotropic phenazine-minus mutations appear to inactivate novel components of the quorum-sensing regulatory network, including one regulator (np20) previously shown to be required for virulence in neutropenic mice.
Functional Analysis of theBacillus subtilisZur Regulon
Authors: Gaballa Ahmed , Wang Tao , Ye Rick W. , Helmann John D.
Published: November 2002

TheBacillus subtiliszinc uptake repressor (Zur) regulates genes involved in zinc uptake. We have used DNA microarrays to identify genes that are derepressed in azurmutant. In addition to members of the two previously identified Zur-regulated operons (yciCandycdHI-yceA), we identified two other genes,yciAandyciB, as targets of Zur regulation. Electrophoretic mobility shift experiments demonstrated that all three operons are direct targets of Zur regulation. Zur binds to an ∼28-bp operator upstream of theyciAgene, as judged by DNase I footprinting, and similar operator sites are found preceding each of the previously described target operons,yciCandycdHI-yceA. Analysis of ayciA-lacZfusion indicates that this operon is induced under zinc starvation conditions and derepressed in thezurmutant. Phenotypic analyses suggest that the YciA, YciB, and YciC proteins may function as part of the same Zn(II) transport pathway. Mutation ofyciAoryciC, singly or in combination, had little effect on growth of the wild-type strain but significantly impaired the growth of theycdHmutant under conditions of zinc limitation. Since the YciA, YciB, and YciC proteins are not obviously related to any known transporter family, they may define a new class of metal ion uptake system. Mutant strains lacking all three identified zinc uptake systems (yciABC,ycdHI-yceA, andzosA) are dependent on micromolar levels of added zinc for optimal growth.
Mutants of
Streptomyces clavuligerus
with Disruptions in Different Genes for Clavulanic Acid Biosynthesis Produce Large Amounts of Holomycin: Possible Cross-Regulation of Two Unrelated Secondary Metabolic Pathways
Authors: de la Fuente Alvaro , Lorenzana Luis M. , Martín Juan F. , Liras Paloma
Published: November 2002

A
Streptomyces clavuligerus ccaR
::
aph
strain, which has a disruption in the regulatory gene
ccaR
, does not produce cephamycin C or clavulanic acid, but does produce a bioactive compound that was identified as holomycin by high-performance liquid chromatography (HPLC) and infrared and mass spectrometry.
S. clavuligerus
strains with disruptions in different genes of the clavulanic acid pathway fall into three groups with respect to holomycin biosynthesis. (i) Mutants with mutations in the early steps of the pathway blocked in the gene
ceaS
(
pyc
) (encoding carboxyethylarginine synthase),
bls
(encoding a β-lactam synthetase), or open reading frame 6 (ORF6; coding for an acetyltransferase of unknown function) are holomycin nonproducers. (ii) Mutants blocked in the regulatory gene
ccaR
or
claR
or blocked in the last gene of the pathway encoding clavulanic acid reductase (
car
) produce holomycin at higher levels than the wild-type strain. (iii) Mutants with disruption in
cyp
(coding for cytochrome P450), ORF12, and ORF15, genes that appear to be involved in the conversion of clavaminic acid into clavaldehyde or in secretion steps, produce up to 250-fold as much holomycin as the wild-type strain. An assay for holomycin synthetase was developed. This enzyme forms holomycin from holothin by using acetyl coenzyme A as an acetyl group donor. The holomycin synthase activities in the different clavulanic acid mutants correlate well with their production of holomycin.
A Third Transposable Element, IS
Ppu12
, from the Toluene-Xylene Catabolic Plasmid pWW0 of
Pseudomonas putida
mt-2
Authors: Williams Peter A. , Jones Rheinallt M. , Shaw Linda E.
Published: November 2002

A 3,372-bp insertion sequence, IS
Ppu12
, has been identified on the archetypal toluene-xylene TOL catabolic plasmid pWW0 from
Pseudomonas putida
mt-2. The insertion sequence element is located on the plasmid between bases 84397 and 87768 in a region which also contains the termini and transposase genes of the catabolic transposons Tn
4651
and Tn
4653
(A. Greated, L. Lambertson, P. A. Williams, and C. M. Thomas, Environ. Microbiol., in press). IS
Ppu12
has terminal inverted repeats of 24 bp with three mismatches and contains four open reading frames, a
tnpA
homologue and three open reading frames (
lspA
,
orf1
, and
orf2)
of undetermined function. After insertion in vitro of a Km
r
cassette into IS
Ppu12
either in the intergenic region between
orf1
and
orf2
or directly into the
orf1
gene and ligation into a suicide vector, the modified IS
Ppu12
-Km transposes at high frequency, often in multiple copies, into the chromosome of a
P. putida
recipient. Inactivation of
lspA
,
orf1
, and
orf2
by introducing a 7-bp deletion into the 5′ region of each gene had no major effect upon transposition, but a similar mutation of
tnpA
completely eliminated transposition. Analysis of the literature and of strains derived from the chlorobenzoate-degrading
Pseudomonas
sp. strain B13 suggests that the promiscuity of this element has played an important role in the history of plasmid pWW0. Database comparisons and the accompanying paper (A. J. Weightman, A. W. Topping, K. E. Hill, L. L. Lee, K. Sakai, J. H. Slater, and A. W. Thomas, J. Bacteriol. 184:6581-6591, 2002) show that IS
Ppu12
is a transposable element also found in other bacteria.
Transposition of
DEH
, a Broad-Host-Range Transposon Flanked by IS
Ppu12
, in
Pseudomonas putida
Is Associated with Genomic Rearrangements and Dehalogenase Gene Silencing
Authors: Weightman Andrew J. , Topping Andrew W. , Hill Katja E. , Lee Li Ling , Sakai Kenji , Slater J. Howard , Thomas Andrew W.
Published: November 2002

Pseudomonas putida
strain PP3 produces two hydrolytic dehalogenases encoded by
dehI
and
dehII
, which are members of different
deh
gene families. The 9.74-kb
DEH
transposon containing
dehI
and its cognate regulatory gene,

dehR
I

, was isolated from strain PP3 by using the TOL plasmid pWW0.
DEH
was fully sequenced and shown to have a composite transposon structure, within which
dehI
and

dehR
I

were divergently transcribed and were flanked on either side by 3.73-kb identical direct repeats. The flanking repeat unit, designated IS
Ppu12
, had the structure of an insertion sequence in that it was bordered by 24-bp near-perfect inverted repeats and contained four open reading frames (ORFs), one of which was identified as
tnpA
, putatively encoding an ISL
3
family transposase. A putative lipoprotein signal peptidase was encoded by an adjacent ORF,
lspA
, and the others, IS
Ppu12 orf1
and
orf2
, were tentatively identified as a truncated cation efflux transporter gene and a PbrR family regulator gene, respectively. The
orf1
-
orf2
intergenic region contained an exact match with a previously described active, outward-orientated promoter, Pout. Transposition of
DEH
-IS
Ppu12
was investigated by cloning the whole transposon into a suicide plasmid donor, pAWT34, and transferring the construct to various recipients. In this way
DEH
-IS
Ppu12
was shown to transpose in a broad range of
Proteobacteria
. Transposition of IS
Ppu12
independently from
DEH
, and inverse transposition, whereby the vector DNA and IS
Ppu12
inserted into the target genome without the
deh
genes, were also observed to occur at high frequencies in
P. putida
PaW340. Transposition of a second
DEH
-IS
Ppu12
derivative introduced exogenously into
P. putida
PP3 via the suicide donor pAWT50 resulted in silencing of resident
dehI
and
dehII
genes in about 10% of transposition transconjugants and provided a genetic link between transposition of IS
Ppu12
and dehalogenase gene silencing. Database searches identified IS
Ppu12
-related sequences in several bacterial species, predominantly associated with plasmids and xenobiotic degradative genes. The potential role of IS
Ppu12
in gene silencing and activation, as well as the adaptation of bacteria to degrade xenobiotic compounds, is discussed.
Transcriptional Phase Variation of a Type III Restriction-Modification System in
Helicobacter pylori
Authors: de Vries Nicolette , Duinsbergen Dirk , Kuipers Ernst J. , Pot Raymond G. J. , Wiesenekker Patricia , Penn Charles W. , van Vliet Arnoud H. M. , Vandenbroucke-Grauls Christina M. J. E. , Kusters Johannes G.
Published: November 2002

Phase variation is important in bacterial pathogenesis, since it generates antigenic variation for the evasion of immune responses and provides a strategy for quick adaptation to environmental changes. In this study, a
Helicobacter pylori
clone, designated MOD525, was identified that displayed phase-variable
lacZ
expression. The clone contained a transcriptional
lacZ
fusion in a putative type III DNA methyltransferase gene (
mod
, a homolog of the gene JHP1296 of strain J99), organized in an operon-like structure with a putative type III restriction endonuclease gene (
res
, a homolog of the gene JHP1297), located directly upstream of it. This putative type III restriction-modification system was common in
H. pylori
, as it was present in 15 out of 16 clinical isolates. Phase variation of the
mod
gene occurred at the transcriptional level both in clone MOD525 and in the parental
H. pylori
strain 1061. Further analysis showed that the
res
gene also displayed transcriptional phase variation and that it was cotranscribed with the
mod
gene. A homopolymeric cytosine tract (C tract) was present in the 5′ coding region of the
res
gene. Length variation of this C tract caused the
res
open reading frame (ORF) to shift in and out of frame, switching the
res
gene on and off at the translational level. Surprisingly, the presence of an intact
res
ORF was positively correlated with active transcription of the downstream
mod
gene. Moreover, the C tract was required for the occurrence of transcriptional phase variation. Our finding that translation and transcription are linked during phase variation through slipped-strand mispairing is new for
H. pylori
.
The Nitrate Reductase and Nitrite Reductase Operons and the
narT
Gene of
Staphylococcus carnosus
Are Positively Controlled by the Novel Two-Component System NreBC
Authors: Fedtke I. , Kamps A. , Krismer B. , Götz F.
Published: November 2002

In
Staphylococcus carnosus
, the
nreABC
(for nitrogen regulation) genes were identified and shown to link the nitrate reductase operon (
narGHJI
) and the putative nitrate transporter gene
narT
. An
nreABC
deletion mutant, m1, was dramatically affected in nitrate and nitrite reduction and growth. Transcription of
narT
,
narGHJI
, and the nitrite reductase (
nir
) operon was severely reduced even when cells were cultivated anaerobically without nitrate or nitrite.
nreABC
transcripts were detected when cells were grown aerobically or anaerobically with or without nitrate or nitrite. NreA is a GAF domain-containing protein of unknown function. In vivo and in vitro studies showed that NreC is phosphorylated by NreB and that phospho-NreC specifically binds to a GC-rich palindromic sequence to enhance transcription initiation. This binding motif was found at the
narGHJI
,
nir
, and
narT
promoters but not at the
moeB
promoter. NreB is a cytosolic protein with four N-terminal cysteine residues. The second cysteine residue was shown to be important for NreB function. In vitro autophosphorylation of NreB was not affected by nitrate, nitrite, or molybdate. The
nir
promoter activity was iron dependent. The data provide evidence for a global regulatory system important for aerobic and anaerobic metabolism, with NreB and NreC forming a classical two-component system and NreB acting as a sensor protein with oxygen as the effector molecule.
Degradation of a
Caulobacter
Soluble Cytoplasmic Chemoreceptor Is ClpX Dependent
Authors: Potocka Isabel , Thein Melanie , Østerås Magne , Jenal Urs , Alley M. R. K.
Published: November 2002

In order to determine whether ClpXP-mediated proteolysis is a common mechanism used to regulate the chemotaxis machinery during the cell cycle of
Caulobacter crescentus
, we have characterized a soluble cytoplasmic chemoreceptor, McpB. The
mcpB
gene lies adjacent to the major chemotaxis operon, which encodes 12 chemotaxis proteins, including the membrane chemoreceptor McpA. Like McpA, McpB possesses a C-terminal CheBR docking motif and three potential methylation sites, which we suggest are methylated. The McpB protein is degraded via a ClpX-dependent pathway during the swarmer-to-stalked cell transition, and a motif, which is 3 amino acids N-terminal to the McpB CheBR docking site, is required for proteolysis. Analysis of the degradation signal in McpB and McpA reveals a common motif present in the other four chemoreceptors that possess CheBR docking sites. A green fluorescent protein (GFP) fusion bearing 58 amino acids from the C terminus of McpA, which contains this motif, is degraded, suggesting that the C-terminal sequence is sufficient to confer ClpXP protease susceptibility.
Transcriptional Response of
Pasteurella multocida
to Defined Iron Sources
Authors: Paustian Michael L. , May Barbara J. , Cao Dongwei , Boley Daniel , Kapur Vivek
Published: November 2002

Pasteurella multocida
was grown in iron-free chemically defined medium supplemented with hemoglobin, transferrin, ferritin, and ferric citrate as iron sources. Whole-genome DNA microarrays were used to monitor global gene expression over seven time points after the addition of the defined iron source to the medium. This resulted in a set of data containing over 338,000 gene expression observations. On average, 12% of
P. multocida
genes were differentially expressed under any single condition. A majority of these genes encoded
P. multocida
proteins that were involved in either transport and binding or were annotated as hypothetical proteins. Several trends are evident when the data from different iron sources are compared. In general, only two genes (
ptsN
and
sapD
) were expressed at elevated levels under all of the conditions tested. The results also show that genes with increased expression in the presence of hemoglobin did not respond to transferrin or ferritin as an iron source. Correspondingly, genes with increased expression in the transferrin and ferritin experiments were expressed at reduced levels when hemoglobin was supplied as the sole iron source. Finally, the data show that genes that were most responsive to the presence of ferric citrate did not follow a trend similar to that of the other iron sources, suggesting that different pathways respond to inorganic or organic sources of iron in
P. multocida
. Taken together, our results demonstrate that unique subsets of
P. multocida
genes are expressed in response to different iron sources and that many of these genes have yet to be functionally characterized.
Leaderless mRNAs Bind 70S Ribosomes More Strongly than 30S Ribosomal Subunits in
Escherichia coli
Authors: O'Donnell Sean M. , Janssen Gary R.
Published: November 2002

By primer extension inhibition assays, 70S ribosomes bound with higher affinity, or stability, than did 30S subunits to leaderless mRNAs containing AUG or GUG start codons. Addition of translation initiation factors affected ribosome binding to leaderless mRNAs. Our results suggest that translation of leaderless mRNAs might initiate through a pathway involving 70S ribosomes or 30S subunits lacking IF3.
cis
-Acting Sequences of
Bacillus subtilis pyrG
mRNA Essential for Regulation by Antitermination
Authors: Meng Qi , Switzer Robert L.
Published: November 2002

Expression of the
Bacillus subtilis pyrG
gene, which encodes CTP synthetase, is repressed by cytidine nucleotides. Regulation involves a termination-antitermination mechanism acting at a transcription terminator located within the 5′ untranslated
pyrG
leader sequence. Deletion and substitution mutagenesis of a series of
pyrG
′
-lacZ
transcriptional fusions integrated into the
B. subtilis
chromosome demonstrated that only the terminator stem-loop and two specific 4- to 6-nucleotide RNA sequences were required for derepression of
pyrG
by starvation for cytidine nucleotides. The first sequence, GGGC/U, comprises the first four nucleotides at the 5′ end of the
pyrG
transcript, and the second, GCUCCC, forms the first six nucleotides of the 5′ strand of the terminator stem. All of the nucleotides lying between the two required RNA sequences can be deleted without loss of regulation. We propose that an as-yet-unidentified regulatory protein binds to these two RNA segments and prevents termination of transcription in the
pyrG
leader region when intracellular CTP levels are low.
The Viable but Nonculturable State and Starvation Are Different Stress Responses of
Enterococcus faecalis
, as Determined by Proteome Analysis
Authors: Heim Sabina , Del Mar Lleo Maria , Bonato Barbara , Guzman Carlos A. , Canepari Pietro
Published: November 2002

The protein expression patterns of exponentially growing, starved, and viable but nonculturable (VBNC)
Enterococcus faecalis
cells were analyzed to establish whether differences exist between the VBNC state and other stress responses. The results indicate that the protein profile of VBNC cells differs from that of either starved or exponentially growing bacteria. This demonstrates that the VBNC state is a distinct physiological phase within the life cycle of
E. faecalis
, which is activated in response to multiple environmental stresses.