bluuebunny/crossref_metadata_2025 huggingface dataset:
Sub-0.1 μm nitride hard mask open process without precuring the ArF photoresist
Authors: Kim J. , Chae Y. S. , Lee W. S. , Shon J. W. , Kang C. J. , Han W. S. , Moon J. T.
Published: March 2003

A nitride hard mask open process with an ArF photoresist (PR) has been achieved in a dual-frequency capacitively coupled plasma-type reactor to pattern 200 nm high and 90 nm wide nitride lines. It was found that the most serious problems of the hard mask open process with ArF PR are striation and wiggling of the PR. Striation is a serious side roughness of nitride lines that results from the deformation of the top portion of the PR during dry etching. This striation is aggravated by the high ion energy and the low selectivity of the nitride to the PR. Wiggling of the PR is defined as the collapse of the PR resulting from slimming of the ArF PR and deposition of a fluorocarbon polymer during dry etching. The wiggling of the PR is strongly dependent on the conditions of the dry etching, such as chamber pressure, chiller temperature, gas chemistry, and total etch time, all of which determine the temperature of the top surface of the ArF PR during dry etching. With the help of plasma diagnostics and an understanding of the mechanism of the striation and wiggling of the ArF PR, nitride lines of 90 nm width and height of 200 nm are successfully patterned without any precuring of the ArF PR.
Response of a Deciduous Forest to the Mount Pinatubo Eruption: Enhanced Photosynthesis
Authors: Gu Lianhong , Baldocchi Dennis D. , Wofsy Steve C. , Munger J. William , Michalsky Joseph J. , Urbanski Shawn P. , Boden Thomas A.
Published: March 2003

Volcanic aerosols from the 1991 Mount Pinatubo eruption greatly increased diffuse radiation worldwide for the following 2 years. We estimated that this increase in diffuse radiation alone enhanced noontime photosynthesis of a deciduous forest by 23% in 1992 and 8% in 1993 under cloudless conditions. This finding indicates that the aerosol-induced increase in diffuse radiation by the volcano enhanced the terrestrial carbon sink and contributed to the temporary decline in the growth rate of atmospheric carbon dioxide after the eruption.
Climate Sensitivity Uncertainty and the Need for Energy Without CO
2
Emission
Authors: Caldeira Ken , Jain Atul K. , Hoffert Martin I.
Published: March 2003

The UN Framework Convention on Climate Change calls for “stabilization of greenhouse gas concentrations at a level that would prevent dangerous anthropogenic interference with the climate system.” Even if we could determine a “safe” level of interference in the climate system, the sensitivity of global mean temperature to increasing atmospheric CO
2
is known perhaps only to a factor of three or less. Here we show how a factor of three uncertainty in climate sensitivity introduces even greater uncertainty in allowable increases in atmospheric CO
2
concentration and allowable CO
2
emissions. Nevertheless, unless climate sensitivity is low and acceptable amounts of climate change are high, climate stabilization will require a massive transition to CO
2
emission–free energy technologies.
A Genomic View of the Human-
Bacteroides thetaiotaomicron
Symbiosis
Authors: Xu Jian , Bjursell Magnus K. , Himrod Jason , Deng Su , Carmichael Lynn K. , Chiang Herbert C. , Hooper Lora V. , Gordon Jeffrey I.
Published: March 2003

The human gut is colonized with a vast community of indigenous microorganisms that help shape our biology. Here, we present the complete genome sequence of the Gram-negative anaerobe
Bacteroides thetaiotaomicron
, a dominant member of our normal distal intestinal microbiota. Its 4779-member proteome includes an elaborate apparatus for acquiring and hydrolyzing otherwise indigestible dietary polysaccharides and an associated environment-sensing system consisting of a large repertoire of extracytoplasmic function sigma factors and one- and two-component signal transduction systems. These and other expanded paralogous groups shed light on the molecular mechanisms underlying symbiotic host-bacterial relationships in our intestine.
Identification of a Testicular Odorant Receptor Mediating Human Sperm Chemotaxis
Authors: Spehr Marc , Gisselmann Günter , Poplawski Alexandra , Riffell Jeffrey A. , Wetzel Christian H. , Zimmer Richard K. , Hatt Hanns
Published: March 2003

Although it has been known for some time that olfactory receptors (ORs) reside in spermatozoa, the function of these ORs is unknown. Here, we identified, cloned, and functionally expressed a previously undescribed human testicular OR, hOR17-4. With the use of ratiofluorometric imaging, Ca
2+
signals were induced by a small subset of applied chemical stimuli, establishing the molecular receptive fields for the recombinantly expressed receptor in human embryonic kidney (HEK) 293 cells and the native receptor in human spermatozoa. Bourgeonal was a powerful agonist for both recombinant and native receptor types, as well as a strong chemoattractant in subsequent behavioral bioassays. In contrast, undecanal was a potent OR antagonist to bourgeonal and related compounds. Taken together, these results indicate that hOR17-4 functions in human sperm chemotaxis and may be a critical component of the fertilization process.
Role of Mobile DNA in the Evolution of Vancomycin-Resistant
Enterococcus faecalis
Authors: Paulsen I. T. , Banerjei L. , Myers G. S. A. , Nelson K. E. , Seshadri R. , Read T. D. , Fouts D. E. , Eisen J. A. , Gill S. R. , Heidelberg J. F. , Tettelin H. , Dodson R. J. , Umayam L. , Brinkac L. , Beanan M. , Daugherty S. , DeBoy R. T. , Durkin S. , Kolonay J. , Madupu R. , Nelson W. , Vamathevan J. , Tran B. , Upton J. , Hansen T. , Shetty J. , Khouri H. , Utterback T. , Radune D. , Ketchum K. A. , Dougherty B. A. , Fraser C. M.
Published: March 2003

The complete genome sequence of
Enterococcus faecalis
V583, a vancomycin-resistant clinical isolate, revealed that more than a quarter of the genome consists of probable mobile or foreign DNA. One of the predicted mobile elements is a previously unknown
vanB
vancomycin-resistance conjugative transposon. Three plasmids were identified, including two pheromone-sensing conjugative plasmids, one encoding a previously undescribed pheromone inhibitor. The apparent propensity for the incorporation of mobile elements probably contributed to the rapid acquisition and dissemination of drug resistance in the enterococci.
Magma Ascent and the Pressurization of Mount Etna's Volcanic System
Authors: Patanè Domenico , De Gori Pasquale , Chiarabba Claudio , Bonaccorso Alessandro
Published: March 2003

After a period of deflation during the 1991–1993 flank eruption, Mount Etna underwent a rapid inflation. Seismicity and ground deformation show that since 1994, a huge volume of magma intruded beneath the volcano, producing from 1998 onward a series of eruptions at the summit and on the flank of the volcano. The last of these, started on 27 October 2002, is still in progress and can be considered one of the most explosive eruptions of the volcano in recent times. Here we show how geodetic data and seismic deformation, between 1994 and 2001, indicate a radial compression around an axial intrusion, consistent with a repressurization of Mount Etna's plumbing system at a depth of 6 to 15 kilometers, which triggered most of the seismicity and provoked the dilatation of the volcano and the recent explosive eruptive activity.
Microscopic Dynamics of Liquid Aluminum Oxide
Authors: Sinn H. , Glorieux B. , Hennet L. , Alatas A. , Hu M. , Alp E. E. , Bermejo F. J. , Price D. L. , Saboungi M.-L.
Published: March 2003

Collective excitations have been observed in liquid aluminum oxide at high temperatures by combining a containerless sample environment with inelastic x-ray scattering. The excitation spectra show a well-defined triplet peak structure at lower wave vectors
Q
(1 to 6 nanometers
−1
) and a single quasi-elastic peak at higher
Q
. The high-
Q
spectra are well described by kinetic theory. The low-
Q
spectra require a frequency-dependent viscosity and provide previously unknown experimental constraints on the behavior of liquids at the interface between atomistic and continuum theory.
Abrupt Climate Change
Authors: Alley R. B. , Marotzke J. , Nordhaus W. D. , Overpeck J. T. , Peteet D. M. , Pielke R. A. , Pierrehumbert R. T. , Rhines P. B. , Stocker T. F. , Talley L. D. , Wallace J. M.
Published: March 2003

Large, abrupt, and widespread climate changes with major impacts have occurred repeatedly in the past, when the Earth system was forced across thresholds. Although abrupt climate changes can occur for many reasons, it is conceivable that human forcing of climate change is increasing the probability of large, abrupt events. Were such an event to recur, the economic and ecological impacts could be large and potentially serious. Unpredictability exhibited near climate thresholds in simple models shows that some uncertainty will always be associated with projections. In light of these uncertainties, policy-makers should consider expanding research into abrupt climate change, improving monitoring systems, and taking actions designed to enhance the adaptability and resilience of ecosystems and economies.
Magmatic Dike Resonances Inferred from Very-Long-Period Seismic Signals
Authors: Kumagai Hiroyuki , Miyakawa Koji , Negishi Hiroaki , Inoue Hiroshi , Obara Kazushige , Suetsugu Daisuke
Published: March 2003

Very-long-period (VLP) signals showing simple decaying harmonic oscillations with periods near 10 seconds and lasting for about 300 seconds were observed in association with an earthquake swarm that occurred beneath Hachijo Island, Japan. Results from the source-mechanism analysis and waveform simulation based on a fluid-filled crack model consistently point to the resonance of a dike filled with a basaltic magma as the source of the VLP signals. Thus, VLP signals can be used to probe the state of the fluid and dynamic processes within a volcanic system.
Polymer Replicas of Photonic Porous Silicon for Sensing and Drug Delivery Applications
Authors: Li Yang Yang , Cunin Frédérique , Link Jamie R. , Gao Ting , Betts Ronald E. , Reiver Sarah H. , Chin Vicki , Bhatia Sangeeta N. , Sailor Michael J.
Published: March 2003

Elaborate one-dimensional photonic crystals are constructed from a variety of organic and biopolymers, which can be dissolved or melted, by templating the solution-cast or injection-molded materials in porous silicon or porous silicon dioxide multilayer (rugate dielectric mirror) structures. After the removal of the template by chemical dissolution, the polymer castings replicate the photonic features and the nanostructure of the master. We demonstrate that these castings can be used as vapor sensors, as deformable and tunable optical filters, and as self-reporting, bioresorbable materials.
Pyogenic Bacterial Infections in Humans with IRAK-4 Deficiency
Authors: Picard Capucine , Puel Anne , Bonnet Marion , Ku Cheng-Lung , Bustamante Jacinta , Yang Kun , Soudais Claire , Dupuis Stéphanie , Feinberg Jacqueline , Fieschi Claire , Elbim Carole , Hitchcock Remi , Lammas David , Davies Graham , Al-Ghonaium Abdulaziz , Al-Rayes Hassan , Al-Jumaah Sulaiman , Al-Hajjar Sami , Al-Mohsen Ibrahim Zaid , Frayha Husn H. , Rucker Rajivi , Hawn Thomas R. , Aderem Alan , Tufenkeji Haysam , Haraguchi Soichi , Day Noorbibi K. , Good Robert A. , Gougerot-Pocidalo Marie-Anne , Ozinsky Adrian , Casanova Jean-Laurent
Published: March 2003

Members of the Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) superfamily share an intracytoplasmic Toll–IL-1 receptor (TIR) domain, which mediates recruitment of the interleukin-1 receptor–associated kinase (IRAK) complex via TIR-containing adapter molecules. We describe three unrelated children with inherited IRAK-4 deficiency. Their blood and fibroblast cells did not activate nuclear factor κB and mitogen-activated protein kinase (MAPK) and failed to induce downstream cytokines in response to any of the known ligands of TIR-bearing receptors. The otherwise healthy children developed infections caused by pyogenic bacteria. These findings suggest that, in humans, the TIR-IRAK signaling pathway is crucial for protective immunity against specific bacteria but is redundant against most other microorganisms.
The Infrared Spectrum of Al
2
H
6
in Solid Hydrogen
Authors: Andrews Lester , Wang Xuefeng
Published: March 2003

Although many volatile binary boron hydride compounds are known, binary aluminum hydride chemistry is limited to the polymeric (AlH
3
)
n
solid. The reaction of laser-ablated aluminum atoms and pure H
2
during codeposition at 3.5 kelvin, followed by ultraviolet irradiation and annealing to 6.5 kelvin, allows dimerization of the intermediate AlH
3
photolysis product to form Al
2
H
6
. The Al
2
H
6
molecule is identified by seven new infrared absorptions that are accurately predicted by quantum chemical calculations for dibridged Al
2
H
6
, a molecule that is isostructural with diborane.
The Pentacovalent Phosphorus Intermediate of a Phosphoryl Transfer Reaction
Authors: Lahiri Sushmita D. , Zhang Guofeng , Dunaway-Mariano Debra , Allen Karen N.
Published: March 2003

Enzymes provide enormous rate enhancements, unmatched by any other type of catalyst. The stabilization of high-energy states along the reaction coordinate is the crux of the catalytic power of enzymes. We report the atomic-resolution structure of a high-energy reaction intermediate stabilized in the active site of an enzyme. Crystallization of phosphorylated β-phosphoglucomutase in the presence of the Mg(II) cofactor and either of the substrates glucose 1-phosphate or glucose 6-phosphate produced crystals of the enzyme–Mg(II)–glucose 1,6-(bis)phosphate complex, which diffracted x-rays to 1.2 and 1.4 angstroms, respectively. The structure reveals a stabilized pentacovalent phosphorane formed in the phosphoryl transfer from the C(1)O of glucose 1,6-(bis)phosphate to the nucleophilic Asp8 carboxylate.
Science Protecting and Protected
Authors: Nightingale Elena O.
Published: March 2003

Science in the Service of Human Rights
. Richard Pierre Claude. University of Pennsylvania Press, Philadelphia, 2002. 275 pp. $42.50, £30. ISBN 0-8122-3679-3.

Claude discusses the complex paths that have led science and scientists toward the protection of human rights.
Making Sense of Dreaming
Authors: Domhoff G. William
Published: March 2003

Dreaming
An Introduction to the Science of Sleep. J. Allan Hobson. Oxford University Press, New York, 2002. 180 pp. $22, £11.99. ISBN 0-19-280304-2.

Building on findings from basic brain research, lab studies of sleep, clinical studies of sleep and dreams, and his records of his own dreams, Hobson explores the mechanisms and functions of dreams and sleep.
Subunit Oligomerization and Substrate Recognition of the
Escherichia coli
ClpYQ (HslUV) Protease Implicated by In Vivo Protein-Protein Interactions in the Yeast Two-Hybrid System
Authors: Lee Yi-Ying , Chang Chiung-Fang , Kuo Chueh-Ling , Chen Meng-Ching , Yu Chien Hung , Lin Pei-I , Wu Whi Fin
Published: April 2003

The
Escherichia coli
ClpYQ (HslUV) is an ATP-dependent protease that consists of an ATPase large subunit with homology to other Clp family ATPases and a peptidase small subunit related to the proteasomal β-subunits of eukaryotes. Six identical subunits of both ClpY and ClpQ self-assemble into an oligomeric ring, and two rings of each subunit, two ClpQ rings surrounded by single ClpY rings, form a dumbbell shape complex. The ClpYQ protease degrades the cell division inhibitor, SulA, and a positive regulator of capsule transcription, RcsA, as well as RpoH, a heat shock sigma transcription factor. Using the yeast-two hybrid system, we explored the in vivo protein-protein interactions of the individual subunits of the ClpYQ protease involved in self-oligomerization, as well as in recognition of specific substrates. Interactions were detected with ClpQ/ClpQ, ClpQ/ClpY, and ClpY/SulA. No interactions were observed in experiments with ClpY/ClpY, ClpQ/RcsA, and ClpQ/SulA. However, ClpY, lacking domain I (ClpY
ΔI
) was able to interact with itself and with intact ClpY. The C-terminal region of ClpY is important for interaction with other ClpY subunits. The previously defined PDZ-like domains at the C terminus of ClpY, including both D1 and D2, were determined to be indispensable for substrate binding. Various deletion and random point mutants of SulA were also made to verify significant interactions with ClpY. Thus, we demonstrated in vivo hetero- and homointeractions of ClpQ and ClpY molecules, as well as a direct association between ClpY and substrate SulA, thereby supporting previous in vitro biochemical findings.
Control of
rep
Gene Expression in Plasmid pGA1 from
Corynebacterium
glutamicum
Authors: Venkova-Canova Tatiana , Pátek Miroslav , Nešvera Jan
Published: April 2003

The cryptic multicopy plasmid pGA1 (4,826 bp) from
Corynebacterium glutamicum
LP-6 belongs to the fifth group of rolling-circle-replicating plasmids. A determinant, which negatively controls pGA1 replication, was localized in the leader region of the
rep
gene coding for the initiator of plasmid replication. This region, when cloned into the compatible vector pEC6, was found to cause decrease of segregational stability of the pGA1 derivative pKG48. A promoter and a single transcriptional start site were found in the
rep
leader region in orientation opposite to the
rep
gene. These results suggest that a small countertranscribed RNA (ctRNA) (ca. 89 nucleotides in length), which might inhibit translation of pGA1
rep
gene, is formed. Analysis of predicted secondary structure of the pGA1-encoded ctRNA revealed features common with the known ctRNAs in bacteria. Inactivation of the promoter P-ctRNA caused a dramatic increase of copies of the respective plasmid, which proved a negative role of the ctRNA in control of pGA1 copy number. A region between the promoters P
rep
and P-ctRNA with a potential to form secondary structures on both ctRNA and
rep
mRNA was found to cause low activity of the
rep
promoter even when promoter P-ctRNA was deleted. Thus, the sequence within the
rep
leader region itself seems to act, in addition to the ctRNA, as a second regulatory element of a novel type, negatively influencing expression of the pGA1
rep
gene.
Genus-Specific Protein Binding to the Large Clusters of DNA Repeats (Short Regularly Spaced Repeats) Present in
Sulfolobus
Genomes
Authors: Peng Xu , Brügger Kim , Shen Biao , Chen Lanming , She Qunxin , Garrett Roger A.
Published: April 2003

Short regularly spaced repeats (SRSRs) occur in multiple large clusters in archaeal chromosomes and as smaller clusters in some archaeal conjugative plasmids and bacterial chromosomes. The sequence, size, and spacing of the repeats are generally constant within a cluster but vary between clusters. For the crenarchaeon
Sulfolobus solfataricus
P2, the repeats in the genome fall mainly into two closely related sequence families that are arranged in seven clusters containing a total of 441 repeats which constitute ca. 1% of the genome. The
Sulfolobus
conjugative plasmid pNOB8 contains a small cluster of six repeats that are identical in sequence to one of the repeat variants in the
S. solfataricus
chromosome. Repeats from the pNOB8 cluster were amplified and tested for protein binding with cell extracts from
S. solfataricus
. A 17.5-kDa SRSR-binding protein was purified from the cell extracts and sequenced. The protein is N terminally modified and corresponds to SSO454, an open reading frame of previously unassigned function. It binds specifically to DNA fragments carrying double and single repeat sequences, binding on one side of the repeat structure, and producing an opening of the opposite side of the DNA structure. It also recognizes both main families of repeat sequences in
S. solfataricus
. The recombinant protein, expressed in
Escherichia coli
, showed the same binding properties to the SRSR repeat as the native one. The SSO454 protein exhibits a tripartite internal repeat structure which yields a good sequence match with a helix-turn-helix DNA-binding motif. Although this putative motif is shared by other archaeal proteins, orthologs of SSO454 were only detected in species within the
Sulfolobus
genus and in the closely related
Acidianus
genus. We infer that the genus-specific protein induces an opening of the structure at the center of each DNA repeat and thereby produces a binding site for another protein, possibly a more conserved one, in a process that may be essential for higher-order stucturing of the SRSR clusters.
Methionine Regeneration and Aminotransferases in
Bacillus subtilis
,
Bacillus cereus
, and
Bacillus anthracis
Authors: Berger Bradley J. , English Shane , Chan Gene , Knodel Marvin H.
Published: April 2003

The conversion of ketomethiobutyrate to methionine has been previously examined in a number of organisms, wherein the aminotransferases responsible for the reaction have been found to be members of the Ia subfamily (L. C. Berger, J. Wilson, P. Wood, and B. J. Berger, J. Bacteriol. 183:4421-4434, 2001). The genome of
Bacillus subtilis
has been found to contain no subfamily Ia aminotransferase sequences. Instead, the analogous enzymes in
B. subtilis
were found to be members of the If subfamily. These putative aspartate aminotransferases, the
yugH
,
ywfG
,
ykrV
,
aspB
, and
patA
gene products, have been cloned, expressed, and characterized for methionine regeneration activity. Only YkrV was able to convert ketomethiobutyrate to methionine, and it catalyzed the reaction only when glutamine was used as amino donor. In contrast, subcellular homogenates of
B. subtilis
and
Bacillus cereus
utilized leucine, isoleucine, valine, alanine, phenylalanine, and tyrosine as effective amino donors. The two putative branched-chain aminotransferase genes in
B. subtilis
,
ybgE
and
ywaA
, were also cloned, expressed, and characterized. Both gene products effectively transaminated branched-chain amino acids and ketoglutarate, but only YbgE converted ketomethiobutyrate to methionine. The amino donor preference for methionine regeneration by YbgE was found to be leucine, isoleucine, valine, phenylalanine, and tyrosine. The
B. subtilis ybgE
gene is a member of the family III of aminotransferases and falls in a subfamily designated here IIIa. Examination of
B. cereus
and
Bacillus anthracis
genome data found that there were no subfamily IIIa homologues in these organisms. In both
B. cereus
and
B. anthracis
, two putative branched-chain aminotransferases and two putative
d
-amino acid aminotransferases were discovered as members of subfamily IIIb. These four sequences were cloned from
B. cereus
, expressed, and characterized. Only the gene product from the sequence designated Bc-BCAT2 was found to convert ketomethiobutyrate to methionine, with an amino donor preference of leucine, isoleucine, valine, phenylalanine, and tyrosine. The
B. anthracis
homologue of Bc-BCAT2 was also cloned, expressed, and characterized and was found to be identical in activity. The aminooxy compound canaline was found to be an uncompetitive inhibitor of
B. subtilis
YbgE and also inhibited growth of
B. subtilis
and
B. cereus
in culture.
Signal Detection and Target Gene Induction by the CpxRA Two-Component System
Authors: DiGiuseppe Patricia A. , Silhavy Thomas J.
Published: April 2003

The Cpx pathway is a two-component signal transduction system that senses a variety of envelope stresses, including misfolded proteins, and responds by upregulating periplasmic folding and trafficking factors. CpxA resides in the inner membrane and has both kinase and phosphatase activities. CpxR, the response regulator, mediates a response by activating transcription of stress-combative genes. Signal transduction is subject to feedback inhibition via regulon member CpxP and autoamplification. Recently, it was shown that the Cpx pathway is also upregulated when cells adhere to hydrophobic surfaces and that this response is dependent on the outer membrane lipoprotein NlpE. Here we show that while NlpE is required for induction of the Cpx pathway by adhesion, induction by envelope stress and during growth is NlpE independent. We show that while all of the envelope stresses tested induce the Cpx pathway in a manner that is dependent on the periplasmic domain of CpxA, induction during growth is independent of CpxA. Therefore, we propose that the Cpx pathway can sense inducing cues that enter the signaling pathway at three distinct points. Although CpxP is not required for induction of the Cpx pathway, we show that its activity as a negative regulator of CpxA is inactivated by envelope stress. Moreover, the
cpxP
promoter is more inducible than any other regulon member tested. Consistent with these results, we suggest that CpxP performs a second function, most likely that of a chaperone. Finally, we show that two Cpx-regulated genes are differentially upregulated in response to different envelope stresses, suggesting the existence of three stress-responsive systems.
Transcription-Defective
soxR
Mutants of
Escherichia coli
: Isolation and In Vivo Characterization
Authors: Chander Monica , Raducha-Grace Laura , Demple Bruce
Published: April 2003

The
soxRS
regulon protects
Escherichia coli
from superoxide and nitric oxide stress. SoxR protein, a transcription factor that senses oxidative stress via its [2Fe-2S] centers, transduces the signal to the
soxS
promoter to stimulate RNA polymerase. Here we describe 29 mutant alleles of
soxR
that cause defects in the activation of
soxS
transcription in response to paraquat, a superoxide stress agent. Owing to the selection and screen used in their isolation, most of these mutant alleles encode proteins that retained specific binding activity for the
soxS
promoter in vivo. The mutations were found throughout the SoxR polypeptide, although those closer to the N terminus typically exhibited greater defects in DNA binding. The degree of the defect in the transcriptional response to superoxide caused by each mutation was closely paralleled by its impaired response to nitric oxide. This work begins the general identification of the residues in the SoxR polypeptide that are critical for transducing oxidative stress signals into gene activation.
Identification and Characterization of a Mandelamide Hydrolase and an NAD(P)+-Dependent Benzaldehyde Dehydrogenase fromPseudomonas putidaATCC 12633
Authors: McLeish Michael J. , Kneen Malea M. , Gopalakrishna Kota N. , Koo Carolyn W. , Babbitt Patricia C. , Gerlt John A. , Kenyon George L.
Published: April 2003

The enzymes of the mandelate metabolic pathway permitPseudomonas putidaATCC 12633 to utilize either or both enantiomers of mandelate as the sole carbon source. The genes encoding the mandelate pathway were found to lie on a single 10.5-kb restriction fragment. Part of that fragment was shown to contain the genes coding for mandelate racemase, mandelate dehydrogenase, and benzoylformate decarboxylase arranged in an operon. Here we report the sequencing of the remainder of the restriction fragment, which revealed three further open reading frames, denotedmdlX,mdlY, andmdlD. All were transcribed in the opposite direction from the genes of themdlABCoperon. Sequence alignments suggested that the open reading frames encoded a regulatory protein (mdlX), a member of the amidase signature family (mdlY), and an NAD(P)+-dependent dehydrogenase (mdlD). ThemdlYandmdlDgenes were isolated and expressed inEscherichia coli, and the purified gene products were characterized as a mandelamide hydrolase and an NAD(P)+-dependent benzaldehyde dehydrogenase, respectively.
Effects of Overexpression of Nutrient Receptors on Germination of Spores of
Bacillus subtilis
Authors: Cabrera-Martinez Rosa-Martha , Tovar-Rojo Federico , Vepachedu Venkata Ramana , Setlow Peter
Published: April 2003

The rates of germination of
Bacillus subtilis
spores with
l
-alanine were increased markedly, in particular at low
l
-alanine concentrations, by overexpression of the tricistronic
gerA
operon that encodes the spore's germinant receptor for
l
-alanine but not by overexpression of
gerA
operon homologs encoding receptors for other germinants. However, spores with elevated levels of the GerA proteins did not germinate more rapidly in a mixture of asparagine, glucose, fructose, and K
+
(AGFK), a germinant combination that requires the participation of at least the germinant receptors encoded by the tricistronic
gerB
and
gerK
operons. Overexpression of the
gerB
or
gerK
operon or both the
gerB
and
gerK
operons also did not stimulate spore germination in AGFK. Overexpression of a mutant
gerB
operon, termed
gerB
*, that encodes a receptor allowing spore germination in response to either
d
-alanine or
l
-asparagine also caused faster spore germination with these germinants, again with the largest enhancement of spore germination rates at lower germinant concentrations. However, the magnitudes of the increases in the germination rates with
d
-alanine or
l
-asparagine in spores overexpressing
gerB
* were well below the increases in the spore's levels of the GerBA protein. Germination of
gerB
* spores with
d
-alanine or
l
-asparagine did not require participation of the products of the
gerK
operon, but germination with these agents was decreased markedly in spores also overexpressing
gerA
. These findings suggest that (i) increases in the levels of germinant receptors that respond to single germinants can increase spore germination rates significantly; (ii) there is some maximum rate of spore germination above which stimulation of GerA operon receptors alone will not further increase the rate of spore germination, as action of some protein other than the germinant receptors can become rate limiting; (iii) while previous work has shown that the wild-type GerB and GerK receptors interact in some fashion to cause spore germination in AGFK, there also appears to be an additional component required for AGFK-triggered spore germination; (iv) activation of the GerB receptor with
d
-alanine or
l
-asparagine can trigger spore germination independently of the GerK receptor; and (v) it is likely that the different germinant receptors interact directly and/or compete with each other for some additional component needed for initiation of spore germination. We also found that very high levels of overexpression of the
gerA
or
gerK
operon (but not the
gerB
or
gerB*
operon) in the forespore blocked sporulation shortly after the engulfment stage, although sporulation appeared normal with the lower levels of
gerA
or
gerK
overexpression that were used to generate spores for analysis of rates of germination.
Bacteriophage SPP1 Chu Is an Alkaline Exonuclease in the SynExo Family of Viral Two-Component Recombinases
Authors: Vellani Trina S. , Myers Richard S.
Published: April 2003

Many DNA viruses concatemerize their genomes as a prerequisite to packaging into capsids. Concatemerization arises from either replication or homologous recombination. Replication is already the target of many antiviral drugs, and viral recombinases are an attractive target for drug design, particularly for combination therapy with replication inhibitors, due to their important supporting role in viral growth. To dissect the molecular mechanisms of viral recombination, we and others previously identified a family of viral nucleases that comprise one component of a conserved, two-component viral recombination system. The nuclease component is related to the exonuclease of phage λ and is common to viruses with linear double-stranded DNA genomes. To test the idea that these viruses have a common strategy for recombination and genome concatemerization, we isolated the previously uncharacterized34.1gene fromBacillus subtilisphage SPP1, expressed it inEscherichia coli, purified the protein, and determined its enzymatic properties. Like λ exonuclease, Chu (the product of34.1) forms an oligomer, is a processive alkaline exonuclease that digests linear double-stranded DNA in a Mg2+-dependent reaction, and shows a preference for 5′-phosphorylated DNA ends. A model for viral recombination, based on the phage λ Red recombination system, is proposed.
Molecular Characterization of the Acid-InducibleasrGene ofEscherichia coliand Its Role in Acid Stress Response
Authors: Šeputienė Vaida , Motiejūnas Domantas , Sužiedėlis Kęstutis , Tomenius Henrik , Normark Staffan , Melefors Öjar , Sužiedėlienė Edita
Published: April 2003

Enterobacteria have developed numerous constitutive and inducible strategies to sense and adapt to an external acidity. These molecular responses require dozens of specific acid shock proteins (ASPs), as shown by genomic and proteomic analysis. Most of the ASPs remain poorly characterized, and their role in the acid response and survival is unknown. We recently identified anEscherichia coligene,asr(acid shock RNA), encoding a protein of unknown function, which is strongly induced by high environmental acidity (pH < 5.0). We show here that Asr is required for growth at moderate acidity (pH 4.5) as well as for the induction of acid tolerance at moderate acidity, as shown by its ability to survive subsequent transfer to extreme acidity (pH 2.0). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of acid-shockedE. colicells harboring a plasmid-borneasrgene demonstrated that the Asr protein is synthesized as a precursor with an apparent molecular mass of 18 kDa. Mutational studies of theasrgene also demonstrated the Asr preprotein contains 102 amino acids. This protein is subjected to an N-terminal cleavage of the signal peptide and a second processing event, yielding 15- and 8-kDa products, respectively. Only the 8-kDa polypeptide was detected in acid-shocked cells containing only the chromosomal copy of theasrgene. N-terminal sequencing and site-directed mutagenesis revealed the two processing sites in the Asr protein precursor. Deletion of amino acids encompassing the processing site required for release of the 8-kDa protein resulted in an acid-sensitive phenotype similar to that observed for theasrnull mutant, suggesting that the 8-kDa product plays an important role in the adaptation to acid shock. Analysis of Asr:PhoA fusions demonstrated a periplasmic location for the Asr protein after removal of the signal peptide. Homologues of theasrgene from otherEnterobacteriaceaewere cloned and shown to be induced inE. coliunder acid shock conditions.
Substrate Specificity Classes and the Recognition Signal for
Salmonella
Type III Flagellar Export
Authors: Hirano Takanori , Minamino Tohru , Namba Keiichi , Macnab Robert M.
Published: April 2003

Most flagellar proteins of
Salmonella
are exported to their assembly destination via a specialized apparatus. This apparatus is a member of the type III superfamily, which is widely used for secretion of virulence factors by pathogenic bacteria. Extensive studies have been carried out on the export of several of the flagellar proteins, most notably the hook protein (FlgE), the hook-capping protein (FlgD), and the filament protein flagellin (FliC). This has led to the concept of two export specificity classes, the rod/hook type and the filament type. However, little direct experimental evidence has been available on the export properties of the basal-body rod proteins (FlgB, FlgC, FlgF, and FlgG), the putative MS ring-rod junction protein (FliE), or the muramidase and putative rod-capping protein (FlgJ). In this study, we have measured the amounts of these proteins exported before and after hook completion. Their amounts in the culture supernatant from a
flgE
mutant (which is still at the hook-type specificity stage) were much higher than those from a
flgK
mutant (which has advanced to the filament-type specificity stage), placing them in the same class as the hook-type proteins. Overproduction of FliE, FlgB, FlgC, FlgF, FlgG, or FlgJ caused inhibition of the motility of wild-type cells and inhibition of the export of the hook-capping protein FlgD. We also examined the question of whether export and translation are linked and found that all substrates tested could be exported after protein synthesis had been blocked by spectinomycin or chloramphenicol. We conclude that the amino acid sequence of these proteins suffices to mediate their recognition and export.
Geobacter sulfurreducensHas Two AutoregulatedlexAGenes Whose Products Do Not Bind therecAPromoter: Differing Responses oflexAandrecAto DNA Damage
Authors: Jara Mónica , Núñuz Cinthia , Campoy Susana , Fernández de Henestrosa Antonio R. , Lovley Derek R. , Barbé Jordi
Published: April 2003

TheEscherichia coliLexA protein was used as a query sequence in TBLASTN searches to identify thelexAgene of the δ-proteobacteriumGeobacter sulfurreducensfrom its genome sequence. The results of the search indicated thatG. sulfurreducenshas two independentlexAgenes designatedlexA1andlexA2. A copy of adinBgene homologue, which inE. coliencodes DNA polymerase IV, is present downstream of eachlexAgene. Reverse transcription-PCR analyses demonstrated that, in both cases,lexAanddinBconstitute a single transcriptional unit. Electrophoretic mobility shift assays with purified LexA1 and LexA2 proteins have shown that both proteins bind the imperfect palindrome GGTTN2CN4GN3ACC found in the promoter region of bothlexA1andlexA2. This sequence is also present upstream of theGeobacter metallireducens lexAgene, indicating that it is the LexA box of this bacterial genus. This palindrome is not found upstream of either theG. sulfurreducensor theG. metallireducens recAgenes. Furthermore, DNA damage induces expression of thelexA-dinBtranscriptional unit but not that of therecAgene. However, the basal level ofrecAgene expression is dramatically higher than that of thelexAgene. Likewise, the promoters of theG. sulfurreducens recN,ruvAB,ssb,umuDC,uvrA, anduvrBgenes do not contain the LexA box and are not likely to bind to the LexA1 or LexA2 proteins.G. sulfurreducensis the first bacterial species harboring alexAgene for which a constitutive expression of itsrecAgene has been described.
Membrane Topology of PssT, the Transmembrane Protein Component of the Type I Exopolysaccharide Transport System in
Rhizobium leguminosarum
bv. trifolii Strain TA1
Authors: Mazur Andrzej , Król Jarosław E. , Marczak Małgorzata , Skorupska Anna
Published: April 2003

The
pssT
gene was identified as the fourth gene located upstream of the
pssNOP
gene cluster possibly involved in the biosynthesis, polymerization, and transport of exopolysaccharide (EPS) in
Rhizobium leguminosarum
bv. trifolii strain TA1. The hydropathy profile and homology searches indicated that PssT belongs to the polysaccharide-specific transport family of proteins, a component of the type I system of the polysaccharide transport. The predicted membrane topology of the PssT protein was examined with a series of PssT-PhoA fusion proteins and a complementary set of PssT-LacZ fusions. The results generally support a predicted topological model for PssT consisting of 12 transmembrane segments, with amino and carboxyl termini located in the cytoplasm. A mutant lacking the C-terminal part of PssT produced increased amounts of total EPS with an altered distribution of high- and low-molecular-weight forms in comparison to the wild-type RtTA1 strain. The PssT mutant produced an increased number of nitrogen fixing nodules on clover.
Regulation of the Alternative Sigma Factor σ
E
during Initiation, Adaptation, and Shutoff of the Extracytoplasmic Heat Shock Response in
Escherichia coli
Authors: Ades Sarah E. , Grigorova Irina L. , Gross Carol A.
Published: April 2003

The alternative sigma factor σ
E
is activated in response to stress in the extracytoplasmic compartment of
Escherichia coli
. Here we show that σ
E
activity increases upon initiation of the stress response by a shift to an elevated temperature (43°C) and remains at that level for the duration of the stress. When the stress is removed by a temperature downshift, σ
E
activity is strongly repressed and then slowly returns to levels seen in unstressed cells. We provide evidence that information about the state of the cell envelope is communicated to σ
E
primarily through the regulated proteolysis of the inner membrane anti-sigma factor RseA, as the degradation rate of RseA is correlated with the changes in σ
E
activity throughout the stress response. However, the relationship between σ
E
activity and the rate of degradation of RseA is complex, indicating that other factors may cooperate with RseA and serve to fine-tune the response.
Transcription of
Clostridium cellulovorans
Cellulosomal Cellulase and Hemicellulase Genes
Authors: Han Sung Ok , Yukawa Hideaki , Inui Masayuki , Doi Roy H.
Published: April 2003

Transcription of the cellulosomal cellulase/hemicellulase genes of
Clostridium cellulovorans
has been investigated by Northern blot, reverse transcriptase PCR (RT-PCR), primer extension, and S1 nuclease analysis. Northern hybridizations revealed that the cellulosomal
cbpA
gene cluster is transcribed as polycistronic mRNAs of 8 and 12 kb. The 8-kb mRNA coded for
cbpA
and
exgS
, and the 12-kb mRNA coded for
cbpA
,
exgS
,
engH
, and
engK
. The sizes of the mRNAs were about 3 kb for
engE
, 1.8 kb for
manA
, 2.7 kb for
xynA
, and 4 kb for
pelA
, indicating monocistronic transcription of these genes. Primer extension and S1 nuclease analysis of
C. cellulovorans
RNA showed that the transcriptional start sites of
cbpA
,
engE
,
manA
, and
hbpA
were located 233, 97, 64, and 61 bp upstream from the first nucleotide of each of the respective translation initiation codons. Alignment of the
cbpA
,
engE
,
manA
, and
hbpA
promoter regions provided evidence for highly conserved sequences that exhibited strong similarity to the σ
A
consensus promoter sequences of gram-positive bacteria.
Porphyrin-Mediated Cell Surface Heme Capture from Hemoglobin byPorphyromonas gingivalis
Authors: Paramaesvaran Mayuri , Nguyen Ky-Anh , Caldon Elizabeth , McDonald James A. , Najdi Sherean , Gonzaga Graciel , Langley David B. , DeCarlo Arthur , Crossley Maxwell J. , Hunter Neil , Collyer Charles A.
Published: April 2003

The porphyrin requirements for growth recovery ofPorphyromonas gingivalisin heme-depleted cultures are investigated. In addition to physiologically relevant sources of heme, growth recovery is stimulated by a number of noniron porphyrins. These data demonstrate that, as forHaemophilus influenzae, reliance on captured iron and on exogenous porphyrin is manifest as an absolute growth requirement for heme. A number of outer membrane proteins including some gingipains contain the hemoglobin receptor (HA2) domain. In cell surface extracts, polypeptides derived from HA2-containing proteins predominated in hemoglobin binding. The in vitro porphyrin-binding properties of a recombinant HA2 domain were investigated and found to be iron independent. Porphyrins that differ from protoporphyrin IX in only the vinyl aspect of the tetrapyrrole ring show comparable effects in competing with hemoglobin for HA2 and facilitate growth recovery. For some porphyrins which differ from protoporphyrin IX at both propionic acid side chains, the modification is detrimental in both these assays. Correlations of porphyrin competition and growth recovery imply that the HA2 domain acts as a high-affinity hemophore at the cell surface to capture porphyrin from hemoglobin. While some proteins involved with heme capture bind directly to the iron center, the HA2 domain ofP. gingivalisrecognizes heme by a mechanism that is solely porphyrin mediated.
Homologue of Macrophage-Activating Lipoprotein in
Mycoplasma
gallisepticum
Is Not Essential for Growth and Pathogenicity in Tracheal Organ Cultures
Authors: Markham Philip F. , Kanci Anna , Czifra György , Sundquist Bo , Hains Peter , Browning Glenn F.
Published: April 2003

While the genomes of a number of
Mycoplasma
species have been fully determined, there has been limited characterization of which genes are essential. The surface protein (p47) identified by monoclonal antibody B3 is the basis for an enzyme-linked immunosorbent assay for serological detection of
Mycoplasma
gallisepticum
infection and appears to be constitutively expressed. Its gene was cloned, and the DNA sequence was determined. Subsequent analysis of the p47 amino acid sequence and searches of DNA databases found homologous gene sequences in the genomes of
M
.
pneumoniae
and
M
.
genitalium
and identity with a gene family in
Ureaplasma
urealyticum
and genes in
M
.
agalactiae
and
M
.
fermentans
. The proteins encoded by these genes were found to belong to a family of basic membrane proteins (BMP) that are found in a wide range of bacteria, including a number of pathogens. Several of the BMP family members, including p47, contain selective lipoprotein-associated motifs that are found in macrophage-activating lipoprotein 404 of
M
.
fermentans
and lipoprotein P48 of
M
.
agalactiae
. The
p47
gene was predicted to encode a 59-kDa peptide, but affinity-purified p47 had a molecular mass of approximately 47 kDa, as determined by polyacrylamide gel analysis. Analysis of native and recombinant p47 by mass peptide fingerprinting revealed the absence of the carboxyl end of the protein encoded by the
p47
gene in native p47, which would account for the difference seen in the predicted and measured molecular weights and indicated posttranslational cleavage of the lipoprotein at its carboxyl end. A DNA construct containing the
p47
gene interrupted by the gene encoding tetracycline resistance was used to transform
M
.
gallisepticum
cells. A tetracycline-resistant mycoplasma clone, P2, contained the construct inserted within the genomic
p47
gene, with crossovers occurring between 73 bp upstream and 304 bp downstream of the inserted tetracycline resistance gene. The absence of p47 protein in clone P2 was determined by the lack of reactivity with rabbit anti-p47 sera or monoclonal antibody B3 in Western blots of whole-cell proteins. There was no difference between the p47
−
mutant and wild-type
M
.
gallisepticum
in pathogenicity in chicken tracheal organ cultures. Thus,
p47
, although homologous to genes that occur in many prokaryotes, is not essential for growth in vitro or for attachment and the initial stages of pathogenesis in chickens.
Function and Regulation of the Formate Dehydrogenase Genes of the Methanogenic ArchaeonMethanococcus maripaludis
Authors: Wood Gwendolyn E. , Haydock Andrew K. , Leigh John A.
Published: April 2003

Methanococcus maripaludisis a mesophilic species ofArchaeacapable of producing methane from two substrates: hydrogen plus carbon dioxide and formate. To study the latter, we identified the formate dehydrogenase genes ofM. maripaludisand found that the genome contains two gene clusters important for formate utilization. Phylogenetic analysis suggested that the two formate dehydrogenase gene sets arose from duplication events within the methanococcal lineage. The first gene cluster encodes homologs of formate dehydrogenase α (FdhA) and β (FdhB) subunits and a putative formate transporter (FdhC) as well as a carbonic anhydrase analog. The second gene cluster encodes only FdhA and FdhB homologs. Mutants lacking eitherfdhAgene exhibited a partial growth defect on formate, whereas a double mutant was completely unable to grow on formate as a sole methanogenic substrate. Investigation offdhgene expression revealed that transcription of both gene clusters is controlled by the presence of H2and not by the presence of formate.