bluuebunny/crossref_metadata_2025 huggingface dataset:
Conjugation Criterion for Making Clearance of the Meshed Helical Surfaces1
Authors: Xiao Da Zhun , Gao Yuan , Wang Zhi Qiang , Liu Dian Min
Published: March 2005

The meshing clearance of the helical surfaces under the condition of conjugation is studied in depth in this paper, which is significant to industrial practice as well as scholastic field. A general conjugation criterion for making clearance of the meshed helical surfaces is clearly defined for the first time, which states that the rotation angle of return stroke caused by clearance for all the points in the tooth surface must be identical. Based on this criterion, it is shown that the commonly adopted uniform clearance is not reasonable for all the meshed helical surfaces. For traditional involute helical surfaces, it is intrinsically correct to adopt the uniform clearance. However, for the helical surfaces with complex transverse tooth profiles, such as those applied to the twin screw compressors, it is unreasonable and harmful to do so due to violation of the conjugation criterion, leading to a severe local contact in the tooth surfaces and higher noise level and, hence, damaging the performance of the helical surfaces in application. As a general principle, the “uniform rotation angle” clearance distribution is a unique one, satisfying the conjugation criterion.
An Advanced Method to Correlate Scale Models With Distorted Configurations
Authors: Cho Uichung , Dutson Alan J. , Wood Kristin L. , Crawford Richard H.
Published: March 2005

Functional testing of prototypes is a critical step in the development of many of today’s products. Results of functional tests allow for verification of proper performance before a product is introduced into the market. The advent of rapid prototyping technologies offers engineers the potential to dramatically reduce the prototype-test-verify cycle and get products to market quickly. However, dimensional and material property limitations of rapid prototypes often prevent them from being used for functional testing without the use of similitude methods to correlate measured prototype behavior with predicted product behavior. The traditional similarity method (TSM), which is based on the Buckingham Π theorem, requires that the dimensionless parameters of the prototype and product systems be identical in order to correlate their states and accurately predict product performance. The requirement of identical dimensionless parameters which is inherent in the TSM is often impossible to realize with the limited properties available from rapid prototyping technologies. In order to overcome this limitation, an empirical similarity method (ESM) has been developed. The general concept of the ESM is introduced along with an implementation procedure. Numerical and experimental examples are presented which demonstrate the feasibility and industrial impact of the ESM in the context of product design.
Mobility Analysis of RSSR Mechanisms by Working Volume
Authors: Chung Wen-Yeuan
Published: March 2005

An RSSR mechanism is analyzed by treating the linkage as two manipulators. The mobility region of the input link or the output link is derived from the intersection region of the working volumes generated by two manipulators. It can be found by solving the region bounded by an ellipse and a unit circle. Concise discriminants and straightforward strategy are developed to determine the type for some special RSSR linkages. Grashof’s rule is derived for determining the type of planar four-bar and proved to be the primary criterion for simply skewed four-bar.
Tolerance Synthesis of Mechanisms: A Robust Design Approach
Authors: Caro Ste´phane , Bennis Fouad , Wenger Philippe
Published: March 2005

This paper provides a new robust design method to dimension a mechanism and to synthesize its dimensional tolerances. The general issue is to find a robust mechanism for a given task, and to compute its optimal dimensional tolerances. For that purpose, the developed approach follows two consecutive steps, which are independent and complementary. First, the dimensions of the mechanism are computed by means of an appropriate robustness index, which is used to minimize the sensitivity of its performances to variations. These robust dimensions are obtained independently of the amount of variations, and tolerate globally the largest variations. Thus, knowing the acceptable performance error of the mechanism, the second step aims at computing the optimal dimensional tolerances of the mechanism by means of the new tolerance synthesis method. This method is used to find the best distribution of the error between the dimensions of the mechanism. Two serial manipulators are studied to illustrate the theory.
Equivalent Kinematic Chains of Three Degree-of-Freedom Tripod Mechanisms With Planar-Spherical Bonds
Authors: Huynh Patrick , Herve´ Jacques M.
Published: March 2005

The paper aims to analyze the equivalent kinematic chains of a family of three-degree-of-freedom (3-DOF) tripod mechanisms with planar-spherical bonds in order to determine the platform motions generated by the mechanisms, and then to develop a prototype of a 3-DOF 3-RPS type parallel mechanism, which can be used as a wrist robotic device. After a short introduction to mechanical generators of Lie subgroups of displacement, the mobility formula of a general 3-DOF tripod mechanism based on the modified Gru¨ebler’s criterion is given. Using displacement group theory theorems, the analyzed closed-loop system becomes finally equivalent to three contacts between a rigid assembly of three moving spheres onto three fixed planes. As an application of the above method, a prototype mechanism is designed and fabricated based on the kinematics analysis, the force capability and the simplicity.
Design of Vibration Absorbers for Step Motions and Step Disturbances
Authors: Fortgang Joel , Singhose William
Published: March 2005

Mechanical systems with flexible dynamics often suffer from vibration induced by changes in the reference command or from external disturbances. The technique of adding a vibration absorber has proven useful at eliminating vibrations from external disturbances and rotational imbalances. Traditionally, vibration absorbers have been designed for systems subject to sinusoidal or random excitations. Here the applicability of vibration absorbers to systems with steplike changes in the reference command or similar disturbances is studied. This type of motion is more common in robotic applications. Here absorbers are designed using two methods; the first technique uses a weighting on peak overshoot and settling time to allow tradeoffs between the two performance criteria. The second simpler method utilizes an eigenvalue technique to predict the time constant. Both of these techniques provide the possibility of significant improvement in settling time. The performance of this absorber design strategies is compared with previously proposed vibration absorbers and experimental results verify its utility.
The Influence of Pulley Deformations on the Shifting Mechanism of Metal Belt CVT
Authors: Carbone G. , Mangialardi L. , Mantriota G.
Published: March 2005

This paper is concerned with the shifting behavior of a metal belt CVT. The calculations are performed for the chain belt case by using a one-dimensional model of the belt: the radial thickness of the belt is neglected. The friction forces are modeled on the basis of the Coulomb friction hypothesis. The deformation of the belt, i.e., the variation of its transversal width, is shown to be negligible with respect to the variation of the local groove width caused by the elastic deformation of the pulleys and by the clearance in the bearings. The particular shape of the deformed pulley is described on the basis of Sattler model (1999) who showed that the variation of the groove angle and that one of the local groove width of the pulley can be easily described by simple trigonometric formulas. The paper shows that the characteristic behavior of the transmission during slow shifting maneuvers, referred to as “creep mode,” is caused by the bending of the pulleys, that is to say for rigid pulleys no “creep mode” can be observed. Moreover, the model shows that increasing the rate of change of speed ratio a transition from the “creep-mode” to the so called “slip-mode” behavior of the variator takes place, as experimentally observed.
Constant Power Continuously Variable Transmission (CP-CVT): Operating Principle and Analysis
Authors: Cretu O. S. , Glovnea R. P.
Published: March 2005

The paper is the first of a series of papers that present an original constant power continuously variable transmission (CP-CVT) traction drive. This paper presents the basic functional principle and demonstrates the device’s characteristics of constant power. The device belongs to the well-known toroidal traction drive family. It comprises of two input discs, one conical and the other toroidal, a conical output disc, and a number of spherical balls. The rotation axis of each ball is self-adjusted according to its geometrical position relative to the input and output discs. A variation of the output torque makes the balls change their position relative to the discs and thus causes a change of the transmission ratio. The kinematics and dynamics of the balls are first performed and then the formulas that relate the power transmitted to the kinematics and geometric parameters are deducted. Finally it is concluded that the CP-CVT presented offers good functional steady power characteristics that could fit the automobile industry requirements.
Selection and Design of Planetary Gear Trains Based on Power Flow Maps
Authors: Salgado David R. , Del Castillo J. M.
Published: March 2005

It is shown that for any planetary gear train (PGT) it is possible to find the relationship existing between the transmission ratio of the train and the power transmitted through each gear pair (gearing power). Specifically, for trains of five and six members, this functional dependence generates a family of curves parameterized by the transmission ratio. A plot of this family of curves is called a power flow map. It allows one to identify those trains that have a potentially high efficiency. In particular, it identifies the transmissions which present power recirculation, a condition that is generally undesirable. Also, an analysis of the power flow maps for the six-member trains leads to the conclusion that, for most of these trains, there is a single expression for the efficiency. The interest of the proposed method is that it is only necessary to know the expression for the PGT’s transmission ratio in order to obtain its power flow map. Also, the map is independent of the constructional solution that is adopted for the train. Finally, the power maps that are obtained are used to find alternatives to the designs of a train consisting of several stages of a basic four-member PGT.
Analysis and Optimization of the Loaded Meshing of Face Gears
Authors: Guingand M. , de Vaujany J. P. , Icard Y.
Published: March 2005

This paper presents a method of simulating loaded face gear meshing. A brief description of the software developed is given in the first part. It simulates the loaded behavior of face gear meshing and provides information such as the instantaneous pressure distribution across the entire width of the teeth in contact and loaded transmission error. Analytical simulations are used to define the geometry and study the unloaded kinematics. This method permits very precise results in a very short time. In the second part of the paper, a comparison between point and line contacts is made for loaded meshing. Further computations calculate assembling-error sensitivity for the two types of contact. The best performance is achieved with the line contact, but it is very sensitive to misalignment. The last part of the paper presents a method for pinion crowning to improve optimization of the line contact case. The aim is to obtain the best loading of face gear meshing in order to avoid line contact sensitivity due to misalignment.
A DH-Parameter Based Condition for 3R Orthogonal Manipulators to Have Four Distinct Inverse Kinematic Solutions
Authors: Wenger P. , Chablat D. , Baili M.
Published: March 2005

Positioning 3R manipulators may have two or four inverse kinematic solutions (IKS). This paper derives a necessary and sufficient condition for 3R positioning manipulators with orthogonal joint axes to have four distinct IKS. We show that the transition between manipulators with two and four IKS is defined by the set of manipulators with a quadruple root of their inverse kinematics. The resulting condition is explicit and states that the last link length of the manipulator must be greater than a quantity that depends on three of its remaining DH parameters. This result is of interest for the design of new manipulators.
Optimal Calibration of Parallel Kinematic Machines
Authors: Verner Marcel , Xi Fengfeng , Mechefske Chris
Published: March 2005

In this paper, a new method for optimal calibration of parallel kinematic machines (PKMs) is presented. The basis of the methodology is to exploit the least error sensitive regions in the workspace to yield optimal calibration. To do so, an error model is developed that takes into consideration all the geometric errors due to imprecision in manufacturing and assembly. Based on this error model, it is shown that the error mapping from the geometric errors to the pose error of the PKM depends on the Jacobian inverse. The Jacobian inverse would introduce spurious errors that would affect the calibration results, if used without proper care. Hence, areas in the workspace with smaller condition numbers are selected for calibration. Simulations and experiments are presented to show the effectiveness of the proposed method. Calibration software based on the proposed method has been embedded in the tripod developed at the National Research Council of Canada’s Integrated Manufacturing Technologies Institute.
Effects of Tryptophan Starvation on Levels of the
trp
RNA-Binding Attenuation Protein (TRAP) and Anti-TRAP Regulatory Protein and Their Influence on
trp
Operon Expression in
Bacillus subtilis
Authors: Yang Wen-Jen , Yanofsky Charles
Published: March 2005

The anti-TRAP protein (AT), encoded by the
rtpA
gene of
Bacillus subtilis
, can bind to and inhibit the tryptophan-activated
trp
RNA-binding attenuation protein (TRAP). AT binding can prevent TRAP from promoting transcription termination in the leader region of the
trp
operon, thereby increasing
trp
operon expression. We show here that AT levels continue to increase as tryptophan starvation becomes more severe, whereas the TRAP level remains relatively constant and independent of tryptophan starvation. Assuming that the functional form of AT is a trimer, we estimate that the ratios of AT trimers per TRAP molecule are 0.39 when the cells are grown under mild tryptophan starvation conditions, 0.83 under more severe starvation conditions, and approximately 2.0 when AT is expressed maximally. As the AT level is increased, a corresponding increase is observed in the anthranilate synthase level. When AT is expressed maximally, the anthranilate synthase level is about 70% of the level observed in a strain lacking TRAP. In a nutritional shift experiment where excess phenylalanine and tyrosine could potentially starve cells of tryptophan, both the AT level and anthranilate synthase activity were observed to increase. Expression of the
trp
operon is clearly influenced by the level of AT.
Characterization of Novel
Mycobacterium tuberculosis
and
Mycobacterium smegmatis
Mutants Hypersusceptible to β-Lactam Antibiotics
Authors: Flores Anthony R. , Parsons Linda M. , Pavelka Martin S.
Published: March 2005

Our laboratory previously constructed mutants of
Mycobacterium tuberculosis
and
Mycobacterium smegmatis
with deletions in the genes for their major β-lactamases, BlaC and BlaS, respectively, and showed that the mutants have increased susceptibilities to most β-lactam antibiotics, particularly the penicillins. However, there is still a basal level of resistance in the mutants to certain penicillins, and the susceptibilities of the mutants to some cephalosporin-based β-lactams are essentially the same as those of the wild types. We hypothesized that characterizing additional mutants (derived from β-lactamase deletion mutants) that are hypersusceptible to β-lactam antibiotics might reveal novel genes involved with other mechanisms of β-lactam resistance, peptidoglycan assembly, and cell envelope physiology. We report here the isolation and characterization of nine β-lactam antibiotic-hypersusceptible transposon mutants, two of which have insertions in genes known to be involved with peptidoglycan biosynthesis (
ponA2
and
dapB
); the other seven mutants have insertions which affect novel genes. These genes can be classified into three groups: those involved with peptidoglycan biosynthesis, cell division, and other cell envelope processes. Two of the peptidoglycan-biosynthetic genes (
ponA2
and
pbpX
) may encode β-lactam antibiotic-resistant enzymes proposed to be involved with the synthesis of the unusual diaminopimelyl linkages within the mycobacterial peptidoglycan.
Percolation of the Phd Repressor-Operator Interface
Authors: Zhao Xueyan , Magnuson Roy David
Published: March 2005

Transcription of the P1 plasmid addiction operon, a prototypical toxin-antitoxin system, is negatively autoregulated by the products of the operon. The Phd repressor-antitoxin protein binds to 8-bp palindromic Phd-binding sites in the promoter region and thereby represses transcription. The toxin, Doc, mediates cooperative interactions between adjacent Phd-binding sites and thereby enhances repression. Here, we describe a homologous operon from
Salmonella enterica
serovar Typhimurium which has the same pattern of regulation but an altered repressor-operator specificity. This difference in specificity maps to the seventh amino acid of the repressor and to the symmetric first and eighth positions of the corresponding palindromic repressor-binding sites. Thus, the repressor-operator interface has coevolved so as to retain the interaction while altering the specificity. Within an alignment of homologous repressors, the seventh amino acid of the repressor is highly variable, indicating that evolutionary changes in repressor specificity may be common in this protein family. We suggest that the robust properties of the negative feedback loop, the fuzzy recognition in the operator-repressor interface, and the duplication and divergence of the repressor-binding sites have facilitated the speciation of this repressor-operator interface. These three features may allow the repressor-operator system to percolate within a nearly neutral network of single-step mutations without the necessity of invoking simultaneous mutations, low-fitness intermediates, or other improbable or rate-limiting mechanisms.
Helical Disposition of Proteins and Lipopolysaccharide in the Outer Membrane ofEscherichia coli
Authors: Ghosh Anindya S. , Young Kevin D.
Published: March 2005

In bacteria, several physiological processes once thought to be the products of uniformly dispersed reactions are now known to be highly asymmetric, with some exhibiting interesting geometric localizations. In particular, the cell envelope ofEscherichia colidisplays a form of subcellular differentiation in which peptidoglycan and outer membrane proteins at the cell poles remain stable for generations while material in the lateral walls is diluted by growth and turnover. To determine if material in the side walls was organized in any way, we labeled outer membrane proteins with succinimidyl ester-linked fluorescent dyes and then grew the stained cells in the absence of dye. Labeled proteins were not evenly dispersed in the envelope but instead appeared as helical ribbons that wrapped around the outside of the cell. By staining the O8 surface antigen ofE. coli2443 with a fluorescent derivative of concanavalin A, we observed a similar helical organization for the lipopolysaccharide (LPS) component of the outer membrane. Fluorescence recovery after photobleaching indicated that some of the outer membrane proteins remained freely diffusible in the side walls and could also diffuse into polar domains. On the other hand, the LPS O antigen was virtually immobile. Thus, the outer membrane ofE. colihas a defined in vivo organization in which a subfraction of proteins and LPS are embedded in stable domains at the poles and along one or more helical ribbons that span the length of this gram-negative rod.
Aminoglycosides Are Captured from both Periplasm and Cytoplasm by the AcrD Multidrug Efflux Transporter of
Escherichia coli
Authors: Aires Julio Ramos , Nikaido Hiroshi
Published: March 2005

To understand better the mechanisms of resistance-nodulation-division (RND)-type multidrug efflux pumps, we examined the
Escherichia coli
AcrD pump, whose typical substrates, aminoglycosides, are not expected to diffuse spontaneously across the lipid bilayer. The hexahistidine-tagged AcrD protein was purified and reconstituted into unilamellar proteoliposomes. Its activity was measured by the proton flux accompanying substrate transport. When the interior of the proteoliposomes was acidified, the addition of aminoglycosides to the external medium stimulated proton efflux and the intravesicular accumulation of radiolabeled gentamicin, suggesting that aminoglycosides can be captured and transported from the external medium in this system (corresponding to cytosol). This activity required the presence of AcrA within the proteoliposomes. Interestingly, the increase in proton efflux also occurred when aminoglycosides were present only in the intravesicular space. This result suggested that AcrD can also capture aminoglycosides from the periplasm to extrude them into the medium in intact cells, acting as a “periplasmic vacuum cleaner.”
SpoIIE Regulates Sporulation but Does Not Directly Affect Solventogenesis in
Clostridium acetobutylicum
ATCC 824
Authors: Scotcher Miles C. , Bennett George N.
Published: March 2005

Using gene expression reporter vectors, we examined the activity of the
spoIIE
promoter in wild-type and
spo0A
-deleted strains of
Clostridium acetobutylicum
ATCC 824. In wild-type cells, the
spoIIE
promoter is active in a transient manner during late solventogenesis, but in strain SKO1, where the sporulation initiator
spo0A
is disrupted, no
spoIIE
promoter activity is detectable at any stage of growth. Strains 824(pMSpo) and 824(pASspo) were created to overexpress
spoIIE
and to decrease
spoIIE
expression via antisense RNA targeted against
spoIIE
, respectively. Some cultures of strains 824(pMSpo) degenerated during fermentations by losing the pSOL1 megaplasmid and hence did not produce the solvents ethanol, acetone, and butanol. The frequent degeneration event was shown to require an intact copy of
spoIIE
. Nondegenerate cultures of 824(pMSpo) exhibited normal growth and solvent production. Strain 824(pASspo) exhibited prolonged solventogenesis characterized by increased production of ethanol (225%), acetone (43%), and butanol (110%). Sporulation in strains harboring pASspo was significantly delayed, with sporulating cells exhibiting altered morphology. These results suggest that SpoIIE has no direct effect on the control of solventogenesis and that the changes in solvent production in
spoIIE
-downregulated cells are mediated by effects on the cell during sporulation.
Menaquinone-Specific Prenyl Reductase from the Hyperthermophilic Archaeon
Archaeoglobus fulgidus
Authors: Hemmi Hisashi , Takahashi Yoshihiro , Shibuya Kyohei , Nakayama Toru , Nishino Tokuzo
Published: March 2005

Four genes that encode the homologues of plant geranylgeranyl reductase were isolated from a hyperthermophilic archaeon
Archaeoglobus fulgidus
, which produces menaquinone with a fully saturated heptaprenyl side chain, menaquinone-7(14H). The recombinant expression of one of the homologues in
Escherichia coli
led to a distinct change in the quinone profile of the host cells, although the homologue is the most distantly related to the geranylgeranyl reductase. The new compounds found in the profile had successively longer elution times than those of ordinary quinones from
E. coli
, i.e., menaquinone-8 and ubiquinone-8, in high-performance liquid chromatography on a reversed-phase column. Structural analyses of the new compounds by electron impact-mass spectrometry indicated that their molecular masses progressively increase relative to the ordinary quinones at a rate of 2 U but that they still contain quinone head structures, strongly suggesting that the compounds are quinones with partially saturated prenyl side chains. In vitro assays with dithionite as the reducing agent showed that the prenyl reductase is highly specific for menaquinone-7, rather than ubiquinone-8 and prenyl diphosphates. This novel enzyme noncovalently binds flavin adenine dinucleotide, similar to geranylgeranyl reductase, but was not able to utilize NAD(P)H as the electron donor, unlike the plant homologue.
Modulation of mRNA Stability Participates in Stationary-Phase-Specific Expression of Ribosome Modulation Factor
Authors: Aiso Toshiko , Yoshida Hideji , Wada Akira , Ohki Reiko
Published: March 2005

The expression of ribosome modulation factor (RMF) is induced during stationary phase in
Escherichia coli
. RMF participates in the dimerization of 70S ribosomes to form the 100S ribosome, which is the translationally inactive form of the ribosome. To elucidate the involvement of the control of mRNA stability in growth-phase-specific
rmf
expression, we investigated
rmf
mRNA stability in stationary-phase cells and cells inoculated into fresh medium. The
rmf
mRNA was found to have an extremely long half-life during stationary phase, whereas destabilization of this mRNA took place after the culture was inoculated into fresh medium. RMF and 100S ribosomes disappeared from cells 1 min after inoculation. In addition to control by ppGpp-dependent transcription, these results indicate that the modulation of
rmf
mRNA stability is also involved in the regulation of growth-phase-specific
rmf
expression. Unexpectedly, the postinoculation degradation of
rmf
mRNA was suppressed by the addition of rifampin, suggesting that de novo RNA synthesis is necessary for degradation. This degradation was also suppressed in both a poly(A) polymerase-deficient and an
rne-131
mutant strain. We cloned and sequenced the 3′-proximal regions of
rmf
mRNAs and found that most of these 3′ ends terminated at the ρ-independent terminator with the addition of a one- to five-A oligo(A) tail in either stationary-phase or inoculated cells. No difference was observed in the length of the poly(A) tail between stationary-phase and inoculated cells. These results suggest that a certain postinoculation-specific regulatory factor participates in the destabilization of
rmf
mRNA and is dependent on polyadenylation.
Function and Expression of an
N
-Acetylneuraminic Acid-Inducible Outer Membrane Channel in
Escherichia coli
Authors: Condemine Guy , Berrier Catherine , Plumbridge Jacqueline , Ghazi Alexandre
Published: March 2005

The
Escherichia coli yjhA
(renamed
nanC)
gene encodes a protein of the KdgM family of outer membrane-specific channels. It is transcribed divergently from
fimB
, a gene involved in the site-specific inversion of the region controlling transcription of the fimbrial structural genes but is separated from it by one of the largest intergenic regions in
E. coli
. We show that
nanC
expression is induced by
N
-acetylneuraminic acid and modulated by
N
-acetylglucosamine. This regulation occurs via the NanR and NagC regulators, which also control
fimB
expression.
nanC
expression is also activated by the regulators cyclic AMP-catabolite activator protein, OmpR, and CpxR. When the NanC protein was reconstituted into liposomes, it formed channels with a conductance of 450 pS at positive potential and 300 to 400 pS at negative potential in 800 mM KCl. The channels had a weak anionic selectivity. In an
ompR
background, where the general porins OmpF and OmpC are absent, NanC is required for growth of
E. coli
on
N
-acetylneuraminic acid as the sole carbon source. All these results suggest that NanC is an
N
-acetylneuraminic acid outer membrane channel protein.
GIL16, a New Gram-Positive Tectiviral Phage Related to theBacillus thuringiensisGIL01 and theBacillus cereuspBClin15 Elements
Authors: Verheust Céline , Fornelos Nadine , Mahillon Jacques
Published: March 2005

One of the most notable characteristics ofTectiviridaeresides in their double-layer coats: the double-stranded DNA is located within a flexible lipoprotein vesicle covered by a rigid protein capsid. Despite their apparent rarity, tectiviruses have an extremely wide distribution compared to other phage groups. Members of this family have been found to infect gram-negative (PRD1 and relatives) as well as gram-positive (Bam35, GIL01, AP50, and φNS11) hosts. Several reports have shown that tectiviruses infecting gram-negative bacteria are closely related, whereas no information is currently available on the genetic relationship among those infecting gram-positive bacteria. The present study reports the sequence of GIL16, a new isolate originating fromBacillus thuringiensis, and a genetic comparison of this isolate with the tectiviral bacteriophages Bam35 and GIL01, which originated fromB. thuringiensisserovars Alesti and Israelensis, respectively. In contrast to PRD1 and its relatives, these are temperate bacteriophages existing as autonomous linear prophages within the host cell. Mutations in a particular motif in both the GIL01 and GIL16 phages are also shown to correlate with a switch to the lytic cycle. Interestingly, both bacterial viruses displayed narrow, yet slightly different, host spectrums. We also explore the hypothesis that pBClin15, a linear plasmid hosted by theBacillus cereusreference strain ATCC 14579, is also a prophage. Sequencing of its inverted repeats at both extremities and a comparison with GIL01 and GIL16 emphasize its relationship to theTectiviridae.
In Vivo Evidence for a
recA
-Independent Recombination Process in
Escherichia coli
That Permits Completion of Replication of DNA Containing UV Damage in Both Strands
Authors: Ozgenc Ali I. , Szekeres Edward S. , Lawrence Christopher W.
Published: March 2005

We have investigated recombination mechanisms promoting the completion of replication in the face of unrepaired DNA damage by transforming an isogenic set of
uvrA6
excision-defective
Escherichia coli
strains with pUC-based plasmids in which each strand carried, at staggered positions, a single thymine-thymine pyrimidine (6-4) pyrimidinone lesion. The distance between the lesions was 28 or 8 bp in one orientation relative to the unidirectional ColE1 origin of replication or, in the other orientation, 30 or 10 bp. C-C mismatches placed opposite each of the T-T photoproducts permit unambiguous detection of the three events that can lead to the completion of replication: sister-strand recombination, translesion replication (TR) on the leading strand, and TR on the lagging strand. We find that
E. coli
possesses a largely constitutive,
recA
-independent sister-strand recombination mechanism that allows 9% or more of these severely compromised plasmids to be fully replicated. In one orientation, such recombination depends partly on
recG
and
priA
but not on
ruvA
,
ruvB
,
ruvC
, or
mutS
and is largely independent of
recF
. In the other orientation, recombination is dependent on none of the genes. The strains used did not contain the cryptic phage encoding
recET
, which encodes enzymes that promote interplasmid recombination. The nature of the
recA
-independent recombination mechanism is not known but could perhaps result from a template-strand-switching, or copy choice, process.
HetR-Dependent and -Independent Expression of Heterocyst-Related Genes in an
Anabaena
Strain Overproducing the NtcA Transcription Factor
Authors: Olmedo-Verd Elvira , Flores Enrique , Herrero Antonia , Muro-Pastor Alicia M.
Published: March 2005

Heterocyst development in the cyanobacterium
Anabaena
sp. strain PCC 7120 depends on both the global nitrogen control transcription factor NtcA and the cell differentiation regulatory protein HetR, with expression of nt
cA
and
hetR
being dependent on each other. In this study we constructed strains that constitutively express the nt
cA
gene leading to high levels of NtcA protein irrespective of the nitrogen source, and we analyzed the effects of such NtcA levels on heterocyst differentiation. In the NtcA-overproducing strain, heterocyst differentiation, induction of NtcA-dependent heterocyst development genes or operons such as
devBCA
or the
cox2
operon, and NtcA-dependent excision of the 11-kb
nifD
-intervening element only took place under nitrogen deficiency. Although functional heterocysts were produced in response to nitrogen step-down, the NtcA overproducing strain could not grow diazotrophically. Overexpression of
ntcA
in a
hetR
background promoted expression of
devBCA
in response to ammonium withdrawal and excision of the 11-kb element even in the presence of combined nitrogen. Our results show that some NtcA-dependent heterocyst-related genes can be expressed independently of HetR.
Functional Domains of NosR, a Novel Transmembrane Iron-Sulfur Flavoprotein Necessary for Nitrous Oxide Respiration
Authors: Wunsch Patrick , Zumft Walter G.
Published: March 2005

Bacterial nitrous oxide (N
2
O) respiration depends on the polytopic membrane protein NosR for the expression of N
2
O reductase from the
nosZ
gene. We constructed His-tagged NosR and purified it from detergent-solubilized membranes of
Pseudomonas stutzeri
ATCC 14405. NosR is an iron-sulfur flavoprotein with redox centers positioned at opposite sides of the cytoplasmic membrane. The flavin cofactor is presumably bound covalently to an invariant threonine residue of the periplasmic domain. NosR also features conserved CX
3
CP motifs, located C-terminally of the transmembrane helices TM4 and TM6. We genetically manipulated
nosR
with respect to these different domains and putative functional centers and expressed recombinant derivatives in a
nosR
null mutant, MK418
nosR
::Tn
5
. NosR's function was studied by its effects on N
2
O respiration, NosZ synthesis, and the properties of purified NosZ proteins. Although all recombinant NosR proteins allowed the synthesis of NosZ, a loss of N
2
O respiration was observed upon deletion of most of the periplasmic domain or of the C-terminal parts beyond TM2 or upon modification of the cysteine residues in a highly conserved motif, CGWLCP, following TM4. Nonetheless, NosZ purified from the recombinant NosR background exhibited in vitro catalytic activity. Certain NosR derivatives caused an increase in NosZ of the spectral contribution from a modified catalytic Cu site. In addition to its role in
nosZ
expression, NosR supports in vivo N
2
O respiration. We also discuss its putative functions in electron donation and redox activation.
Substrate Specificities and Expression Patterns Reflect the Evolutionary Divergence of Maltose ABC Transporters in
Thermotoga maritima
Authors: Nanavati Dhaval M. , Nguyen Tu N. , Noll Kenneth M.
Published: March 2005

Duplication of transporter genes is apparent in the genome sequence of the hyperthermophilic bacterium
Thermotoga maritima
. The physiological impacts of these duplications are not well understood, so we used the bacterium's two putative maltose transporters to begin a study of the evolutionary relationship between a transporter's function and the control of expression of its genes. We show that the substrate binding proteins encoded by these operons, MalE1 and MalE2, have different substrate specificities and affinities and that they are expressed under different growth conditions. MalE1 binds maltose (dissociation constant [

K
D

], 24 ± 1 μM), maltotriose (

K
D

, 8 ± 0.5 nM), and β-(1→4)-mannotetraose (

K
D

, 38 ± 1 μM). In contrast, MalE2 binds maltose (

K
D

, 8.4 ± 1 μM), maltotriose (

K
D

, 11.5 ± 1.5 μM), and trehalose (

K
D

, 9.5 ± 1.0 μM) confirming the findings of Wassenberg et al. (J. Mol. Biol. 295:279-288, 2000). Neither protein binds lactose. We examined the expression of these operons at both the transcriptional and translational levels and found that MalE1 is expressed in cells grown on lactose or guar gum and that MalE2 is highly expressed in starch- and trehalose-grown cells. Evidence is provided that
malE1
,
malF1
, and perhaps
malG1
are cotranscribed and so constitute an operon. An open reading frame encoding a putative transcriptional regulatory protein adjacent to this operon (TM1200) is also up-regulated in response to growth on lactose. These evolutionarily related transporter operons have diverged both in function and expression to assume apparently different physiological roles.
The Rok Protein of
Bacillus subtilis
Represses Genes for Cell Surface and Extracellular Functions
Authors: Albano Mark , Smits Wiep Klaas , Ho Linh T. Y. , Kraigher Barbara , Mandic-Mulec Ines , Kuipers Oscar P. , Dubnau David
Published: March 2005

Rok is a repressor of the transcriptional activator ComK and is therefore an important regulator of competence in
Bacillus subtilis
(T. T. Hoa, P. Tortosa, M. Albano, and D. Dubnau, Mol. Microbiol. 43:15-26, 2002). To address the wider role of Rok in the physiology of
B. subtilis
, we have used a combination of transcriptional profiling, gel shift experiments, and the analysis of
lacZ
fusions. We demonstrate that Rok is a repressor of a family of genes that specify membrane-localized and secreted proteins, including a number of genes that encode products with antibiotic activity. We present evidence for the recent introduction of
rok
into the
B. subtilis-Bacillus licheniformis-Bacilllus amyloliquefaciens
group by horizontal transmission.
Cytochrome
bd
Oxidase, Oxidative Stress, and Dioxygen Tolerance of the Strictly Anaerobic Bacterium
Moorella thermoacetica
Authors: Das Amaresh , Silaghi-Dumitrescu Radu , Ljungdahl Lars G. , Kurtz Donald M.
Published: March 2005

The gram-positive, thermophilic, acetogenic bacterium
Moorella thermoacetica
can reduce CO
2
to acetate via the Wood-Ljungdahl (acetyl coenzyme A synthesis) pathway. This report demonstrates that, despite its classification as a strict anaerobe,
M. thermoacetica
contains a membrane-bound cytochrome
bd
oxidase that can catalyze reduction of low levels of dioxygen. Whole-cell suspensions of
M. thermoacetica
had significant endogenous O
2
uptake activity, and this activity was increased in the presence of methanol or CO, which are substrates in the Wood-Ljungdahl pathway. Cyanide and azide strongly (∼70%) inhibited both the endogenous and CO/methanol-dependent O
2
uptake. UV-visible light absorption and electron paramagnetic resonance spectra of
n
-dodecyl-β-maltoside extracts of
M. thermoacetica
membranes showed the presence of a cytochrome
bd
oxidase complex containing cytochrome
b
561
, cytochrome
b
595
, and cytochrome
d
(chlorin). Subunits I and II of the
bd
oxidase were identified by N-terminal amino acid sequencing. The
M. thermoacetica
cytochrome
bd
oxidase exhibited cyanide-sensitive quinol oxidase activity. The
M. thermoacetica
cytochrome
bd
(
cyd
) operon consists of four genes, encoding subunits I and II along with two ABC-type transporter proteins, homologs of which in other bacteria are required for assembly of the
bd
complex. The level of this
cyd
operon transcript was significantly increased when
M. thermoacetica
was grown in the absence of added reducing agent (cysteine + H
2
S). Expression of a 35-kDa cytosolic protein, identified as a cysteine synthase (CysK), was also induced by the nonreducing growth conditions. The combined evidence indicates that cytochrome
bd
oxidase and cysteine synthase protect against oxidative stress and contribute to the limited dioxygen tolerance of
M. thermoacetica
.
A Tetrahydrofolate-Dependent
O
-Demethylase, LigM, Is Crucial for Catabolism of Vanillate and Syringate in
Sphingomonas paucimobilis
SYK-6
Authors: Abe Tomokuni , Masai Eiji , Miyauchi Keisuke , Katayama Yoshihiro , Fukuda Masao
Published: March 2005

Vanillate and syringate are converted into protocatechuate (PCA) and 3-
O
-methylgallate (3MGA), respectively, by
O
-demethylases in
Sphingomonas paucimobilis
SYK-6. PCA is further degraded via the PCA 4,5-cleavage pathway, while 3MGA is degraded through multiple pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and an unidentified 3MGA
O
-demethylase and gallate dioxygenase are participants. For this study, we isolated a 4.7-kb SmaI fragment that conferred on
Escherichia coli
the activity required for the conversion of vanillate to PCA. The nucleotide sequence of this fragment revealed an open reading frame of 1,413 bp (
ligM
), the deduced amino acid sequence of which showed 49% identity with that of the tetrahydrofolate (H
4
folate)-dependent syringate
O
-demethylase gene (
desA
). The
metF
and
ligH
genes, which are thought to be involved in H
4
folate-mediated C
1
metabolism, were located just downstream of
ligM
. The crude LigM enzyme expressed in
E. coli
converted vanillate and 3MGA to PCA and gallate, respectively, with similar specific activities, and only in the presence of H
4
folate; however, syringate was not a substrate for LigM. The disruption of
ligM
led to significant growth retardation on both vanillate and syringate, indicating that
ligM
is involved in the catabolism of these substrates. The ability of the
ligM
mutant to transform vanillate was markedly decreased, and this mutant completely lost the 3MGA
O
-demethylase activity. A
ligM desA
double mutant completely lost the ability to transform vanillate, thus indicating that
desA
also contributes to vanillate degradation. All of these results indicate that
ligM
encodes vanillate/3MGA
O
-demethylase and plays an important role in the O demethylation of vanillate and 3MGA, respectively.
CpxR/OmpR Interplay Regulates Curli Gene Expression in Response to Osmolarity inEscherichia coli
Authors: Jubelin Gregory , Vianney Anne , Beloin Christophe , Ghigo Jean-Marc , Lazzaroni Jean-Claude , Lejeune Philippe , Dorel Corinne
Published: March 2005

Curli fibers could be described as a virulence factor able to confer adherence properties to both abiotic and eukaryotic surfaces. The ability to adapt rapidly to changing environmental conditions through signal transduction pathways is crucial for the growth and pathogenicity of bacteria. OmpR was shown to activatecsgDexpression, resulting in curli production. The CpxR regulator was shown to negatively affect curli gene expression when binding to its recognition site that overlaps thecsgDOmpR-binding site. This study was undertaken to clarify how the interplay between the two regulatory proteins, OmpR and CpxR, can affect the transcription of the curli gene in response to variation of the medium osmolarity. Band-shift assays with purified CpxR proteins indicate that CpxR binds to thecsgDpromoter region at multiple sites that are ideally positioned to explain thecsgrepression activity of CpxR. To understand the physiological meaning of this in vitro molecular phenomenon, we analyzed the effects of an osmolarity shift on the two-component pathway CpxA/CpxR. We establish here that the Cpx pathway is activated at both transcriptional and posttranscriptional levels in response to a high osmolarity medium and that CpxR repressescsgDexpression in high-salt-content medium, resulting in low curli production. However,csgDrepression in response to high sucrose content is not mediated by CpxR but by the global regulatory protein H-NS. Therefore, multiple systems (EnvZ/OmpR, Cpx, Rcs, and H-NS) appear to be involved in sensing environmental osmolarity, leading to sophisticated regulation of the curli genes.
Nucleotide Sequence of the Head Assembly Gene Cluster of Bacteriophage L and Decoration Protein Characterization
Authors: Gilcrease Eddie B. , Winn-Stapley Danella A. , Hewitt F. Curtis , Joss Lisa , Casjens Sherwood R.
Published: March 2005

The temperate
Salmonella enterica
bacteriophage L is a close relative of the very well studied bacteriophage P22. In this study we show that the L procapsid assembly and DNA packaging genes, which encode terminase, portal, scaffold, and coat proteins, are extremely close relatives of the homologous P22 genes (96.3 to 99.1% identity in encoded amino acid sequence). However, we also identify an L gene,
dec
, which is not present in the P22 genome and which encodes a protein (Dec) that is present on the surface of L virions in about 150 to 180 molecules/virion. We also show that the Dec protein is a trimer in solution and that it binds to P22 virions in numbers similar to those for L virions. Its binding dramatically stabilizes P22 virions against disruption by a magnesium ion chelating agent. Dec protein binds to P22 coat protein shells that have expanded naturally in vivo or by sodium dodecyl sulfate treatment in vitro but does not bind to unexpanded procapsid shells. Finally, analysis of phage L restriction site locations and a number of patches of nucleotide sequence suggest that phages ST64T and L are extremely close relatives, perhaps the two closest relatives that have been independently isolated to date among the lambdoid phages.
Magnesium Promotes Flagellation ofVibrio fischeri
Authors: O'Shea Therese M. , DeLoney-Marino Cindy R. , Shibata Satoshi , Aizawa Shin-Ichi , Wolfe Alan J. , Visick Karen L.
Published: March 2005

The bacteriumVibrio fischerirequires bacterial motility to initiate colonization of the Hawaiian squidEuprymna scolopes. Once colonized, however, the bacterial population becomes largely unflagellated. To understand environmental influences onV. fischerimotility, we investigated migration of this organism in tryptone-based soft agar media supplemented with different salts. We found that optimal migration required divalent cations and, in particular, Mg2+. At concentrations naturally present in seawater, Mg2+improved migration without altering the growth rate of the cells. Transmission electron microscopy and Western blot experiments suggested that Mg2+addition enhanced flagellation, at least in part through an effect on the steady-state levels of flagellin protein.
Cyclic AMP (cAMP) and cAMP Receptor Protein Influence both Synthesis and Uptake of Extracellular Autoinducer 2 inEscherichia coli
Authors: Wang Liang , Hashimoto Yoshifumi , Tsao Chen-Yu , Valdes James J. , Bentley William E.
Published: March 2005

Bacterial autoinducer 2 (AI-2) is proposed to be an interspecies mediator of cell-cell communication that enables cells to operate at the multicellular level. Many environmental stimuli have been shown to affect the extracellular AI-2 levels, carbon sources being among the most important. In this report, we show that both AI-2 synthesis and uptake inEscherichia coliare subject to catabolite repression through the cyclic AMP (cAMP)-CRP complex, which directly stimulates transcription of thelsr(for “luxSregulated”) operon and indirectly repressesluxSexpression. Specifically, cAMP-CRP is shown to bind to a CRP binding site located in the upstream region of thelsrpromoter and works with the LsrR repressor to regulate AI-2 uptake. The functions of thelsroperon and its regulators, LsrR and LsrK, previously reported inSalmonella entericaserovar Typhimurium, are confirmed here forE. coli.The elucidation of cAMP-CRP involvement inE. coliautoinduction impacts many areas, including the growth ofE. coliin fermentation processes.